Abstract:

The present invention includes compositions and methods for treating and
delivering medicinal formulations using an inhaler. The composition
includes a space filled flocculated suspension having one or more
flocculated particles of one or more active agents and a
hydrofluoroalkane propellant. A portion of the one or more flocculated
particles is templated by the formation of hydrofluoroalkane droplets
upon atomization and the templated floc compacts upon the evaporation of
the hydrofluoroalkane propellant to form a porous particle for deep lung
delivery.

Claims:

1. A unit-dose delivery system used as a template for use in a dry powder
inhaler comprising:a unit-dose delivery system comprising one or more
concave indentations;a cover positioned to sealed the one or more concave
indentations; anda brittle matrix medicinal formulation appropriate for
pulmonary delivery in at least one of the one or more concave
indentations, wherein the brittle matrix medicinal formulation comprises
a non-tightly packed porous flocculated web matrix comprising one or more
brittle-matrix particles of one or more active agents, wherein a portion
of the one or more brittle-matrix particles is delivered and templated by
the formation of one or more particles upon atomization from the
unit-dose delivery system using a dry powder inhaler to form a respirable
porous particle for deep lung delivery.

2. The medicinal formulation of claim 1, wherein the one or more active
agents comprise itraconazole, voriconazole, paclitaxel, sirolimus,
cyclosporin, an inhalable medicinally active drug for treatment of
asthma, copd, or interstitial lung disease, mycophenolic acid or a salt
thereof, tacrolimus and lactose, or tacrolimus.

3. The medicinal formulation of claim 1, wherein the one or more active
agents comprise a low molecular weight drug, a high molecular weight
drug, a peptide, a protein or a combination thereof.

4. The medicinal formulation of claim 1, wherein the one or more
brittle-matrix particles comprise particles in the form that enables
delivery of the pharmaceutically active drug to the lung, such as in the
form of rods or plates.

5. The medicinal formulation of claim 1, wherein the one or more
brittle-matrix particles are formed by rapid freezing.

6. The medicinal formulation of claim 1, wherein the one or more
brittle-matrix particles are formed by freezing and lyophilizing directly
in the unit-dose delivery system.

7. The medicinal formulation of claim 1, wherein the unit-dose pack is a
blister pack.

8. The medicinal formulation of claim 1, wherein the brittle matrix
particles have skeletal densities less than about 1 g/mL, less than about
0.1 g/mL, and less than about 0.05 g/mL.

9. The medicinal formulation of claim 1, wherein the brittle matrix
particles have a Carr's index or measure of compressibility of greater
than about 10, greater than about 20, greater than about 35.

10. A medicinal formulation for use in a dry powder inhaler comprising:a
non-tightly packed porous flocculated web composition comprising one or
more brittle-matrix particles of one or more active agents, wherein a
portion of the one or more brittle-matrix particles is templated by a
patient and/or device induced shearing energy to form a porous particle
for deep lung delivery.

11. The medicinal formulation of claim 10, wherein the one or more active
agents comprise itraconazole, voriconazole, paclitaxel, sirolimus,
cyclosporin, an inhalable medicinally active drug for treatment of
asthma, copd, or interstitial lung disease, mycophenolic acid or a salt
thereof, tacrolimus and lactose, or tacrolimus.

12. The medicinal formulation of claim 10, wherein the one or more active
agents comprise a low molecular weight drug, a high molecular weight
drug, a peptide, a protein or a combination thereof.

13. The medicinal formulation of claim 10, wherein the one or more active
agents are selected from a protein, a peptide, a vasoactive peptide, an
immunoglobulin, an immunomodulating protein, a hematopoietic factor,
insulin, an insulin analog, amylin, an antibiotic, an antibody an
antigen, an interleukin, an interferon, an erythropoietin, a heparin, a
thrombolytic, an antitrypsin, an enzyme, an anti-protease, a hormone, a
growth factor, a nucleic acid, an oligonucleotide, an antisense agent and
mixtures thereof

15. The medicinal formulation of claim 10, wherein the one or more
brittle-matrix particles comprise a non-tightly packed porous flocculated
web matrix comprising one or more brittle-matrix particles of one or more
active agents, wherein a portion of the one or more brittle-matrix
particles is delivered and templated by the formation of one or more
particles upon atomization.

16. The medicinal formulation of claim 10, wherein the one or more
brittle-matrix particles are formed by freezing.

17. The medicinal formulation of claim 10, wherein the one or more
brittle-matrix particles are formed by freezing and lyophilizing directly
in the unit-dose delivery system.

19. A method of making a dispersible brittle templated composition for a
dry powder inhaler system comprising the steps of:cooling a unit-dose
delivery system intended for one or more metered doses for
inhalation;depositing one or more drops of a drug solution on the
unit-dose delivery system, wherein the drug solution comprises one or
more active pharmaceutical ingredients, one or more solvents, and one or
more excipients, where said drop freezes upon contact with the packaging
material;lyophilizing the pharmaceutical product to produce a non-tightly
packed brittle matrix;equilibrating the non-tightly packed brittle matrix
to room temperature; andcombining the non-tightly packed brittle matrix
with a suitable dry powder inhalation device.

20. The method of 19, wherein the non-tightly packed brittle matirx
composition comprise particles in the form of a web of interconnected
rods or plates.

21. The method of 19, wherein the non-tightly packed brittle matrix
composition comprise particles in the form of a nanostructured web.

23. The composition of claim 19 wherein active agents may be combined with
a pharmaceutical excipient suitable for inhalation.

24. The composition of claim 19, wherein the one or more brittle matrix
particles of anisotropic particles comprise a low molecular weight drug,
a high molecular weight drug, a peptide, a protein or a combination
thereof.

25. The method of claim 19, wherein the one or more active agents are
selected from a protein, a peptide, a vasoactive peptide, an
immunoglobulin, an immunomodulating protein, a hematopoietic factor,
insulin, an insulin analog, amylin, an antibiotic, an antibody an
antigen, an interleukin, an interferon, an erythropoietin, a heparin, a
thrombolytic, an antitrypsin, an enzyme, an anti-protease, a hormone, a
growth factor, a nucleic acid, an oligonucleotide, an antisense agent,
albumin and mixtures thereof.

26. A drug product produced by the process of claim 19.

27. A method of making a dispersible brittle templated composition for a
dry powder inhaler system comprising the steps of:forming a non-tightly
packed brittle matrix from one or more drops of drug solution comprising
one or more active pharmaceutical ingredients, one or more solvents, and
one or more excipients;lyophilizing the non-tightly packed brittle
matrix;equilibrating the non-tightly packed brittle matrix to room
temperature;portioning the non-tightly packed brittle matrix into sample
doses; andcombining the non-tightly packed brittle matrix with a suitable
dry powder inhalation device.

28. A medicinal formulation for use in a dry powder inhaler comprising:a
non-tightly packed porous flocculated web composition comprising one or
more brittle-matrix particles of one or more active agents, wherein a
portion of the one or more brittle-matrix particles is templated by a
patient and/or device induced shearing energy to form a porous particle
for deep lung delivery.

29. A medicinal formulation of claim 28, wherein the brittle-matrix
powders are delivered in a unit-dose delivery system, such as a blister
pack, a capsule from a single-dose or multiple-dose DPI device or a
reservoir from a multi-dose DPI device.

30. The medicinal formulation of claim 28, wherein the one or more active
agents comprise itraconazole, paclitaxel, tacrolimus and lactose, or
tacrolimus.

31. The medicinal formulation of claim 28, wherein the one or more active
agents comprise a low molecular weight drug, a high molecular weight
drug, a peptide, a protein or a combination thereof.

32. The medicinal formulation of claim 28, wherein the brittle matrix
particles have skeletal densities less than about 1 g/mL, less than about
0.1 g/mL, and less than about 0.05 g/mL.

33. The medicinal formulation of claim 28, wherein the brittle matrix
particles have a Carr's index (measure of compressibility) of greater
than about 10, greater than about 20, greater than about 35.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application is a continuation-in-part and claims priority based
on U.S. patent application Ser. No. 12/371,573, filed Feb. 13, 2009,
which claims priority to U.S. Provisional Application Ser. No.
61/028,218, filed Feb. 13, 2008, the contents of each of which are
incorporated by reference herein in their entireties.

[0004]Without limiting the scope of the invention, its background is
described in connection with medicinal formulations and compositions for
use in pressurized metered dose inhalers. Current methods of delivery
have produced few examples of suspensions with 1-5% (w/w) mass loadings
in HFAs that are stable against settling on time scales of over 60
seconds. As the mass loading increases up to and above 5% (w/w),
particles often aggregate within aerosolized droplets leading to
substantial increases in da and thus reduction in fine particle
fraction (FPF).

[0005]For example, U.S. Pat. No. 6,585,957 relates to medicinal aerosol
formulations. The formulation includes a protein or peptide medicament, a
fluid carrier for containing said medicament; and a stabilizer selected
from an amino acid, a derivative thereof or a mixture of the foregoing.
Similarly, U.S. Pat. No. 6,655,381 relates to pre-metered dose magazine
for breath-actuated dry powder inhaler. More specifically, a pre-metered
dose assembly for consistently supplying precise doses of medicament is
taught for a breath-actuated dry powder inhaler. The breath-actuated dry
powder inhaler including the pre-metered dose assembly in combination
with a de-agglomerator for breaking up aggregates and micronizing
particles of dry powder prior to inhalation of the powder by a patient.

[0006]U.S. Pat. No. 7,011,818 relates to carrier particles for use in dry
powder inhalers. The powder includes additive material on the surfaces of
the carrier particles to promote the release of the active particles from
the carrier particles on actuation of the inhaler. The powder is such
that the active particles are not liable to be release from the carrier
particles before actuation of the inhaler. The inclusion of additive
material (4) in the powder has been found to give an increased respirable
fraction of the active material

[0007]The general method of delivery of drugs to the lungs for the
treatment of numerous pulmonary disorders is through inhalation of the
drug particles. The drug particles are generally in the form of an
aerosol of respirable sized particles incorporated into a colloidal
dispersion containing either a propellant, as a pressurized metered dose
inhaler (pMDI) or air such as is the case with a dry powder inhaler
(DPI).

[0008]It is of the upmost importance in the aerosol formulation that the
composition is stable and the dose discharged from the metered dose valve
is reproducible; however, there are numerous factors that influence these
features, e.g., creaming, or settling, after agitation are common sources
of dose irreproducibility in suspension formulations. Another concern is
the flocculation of the composition after agitation. This flocculation
often results in dose irreproducibility and as such, it is an undesirable
process and composition and is often seen in aerosol formulations
containing only medicament and propellant or formulation contains small
amounts of surfactants. Surfactants are often included in the
formulations to serve as suspending aids to stabilize the suspension or
lubricants to reduce valve sticking which also causes dose
irreproducibility.

[0009]In addition, the drug absorption into the subject from the airway
dependents on numerous factors, e.g., the composition of the formulation,
type of solute, the method of drug delivery, and the site of deposition.
Therefore, formulation and device characteristics have a dramatic impact
upon the rate and extent of peptide absorption from the lung. Dry powder
presentations of peptide and protein drugs possess unique opportunities
in formulations, which do not occur in liquid presentations such as pMDIs
and nebulized solutions.

[0010]One method commonly used to prepare medicament particles for drug
formulations into fine powder is spray drying. Spray drying forms
spherical particles that are often hollow thus resulting in a powder with
low bulk density compared to the initial material, other characteristics
include particle size distribution, bulk density, porosity, moisture
content, dispersibility, etc. In addition, the spray dried particles
demonstrate poor flow characteristics. The spray drying process requires
heating of the formulation making it drying less desirable for heat
sensitive compounds such as peptide and protein drugs. For these reasons
spray dried particles often suffer from adhesion and poor flowability to
the extent that dose accuracy becomes a problem.

SUMMARY OF THE INVENTION

[0011]The present invention provides for the dispensing of poorly water
soluble compositions and/or protein via pMDI. As stated previously,
sub-micron particles are desirable for drug delivery because smaller
particles provide a larger surface area/mass ratio for dissolution.
Milling is a common particle size reduction method; however, the milling
process has been shown to produce partially amorphous drug domains.
Although amorphous particles may be desirable for certain applications
(e.g., to raise solubility for enhanced bioavailability), they are
equally undesirable in many applications (e.g., the drug nanoparticles
may crystallize upon storage). Thus the inventors recognized that it is
important to find ways to make crystalline nanocrystals without the need
to use milling.

[0012]The present invention provides for the formation of stable
suspensions of very low density flocs of rod-shaped drugs in
hydrofluoroalkane propellants for pressurized meter dose inhalers (pMDI)
and for templating the flocs to achieve high fine particle fractions in
pulmonary delivery.

[0013]The present invention also provides a unit-dose delivery system used
as a template for use in a dry powder inhaler. The invention includes a
unit-dose delivery system comprising one or more concave indentations; a
cover positioned to sealed the one or more concave indentations; and a
brittle matrix medicinal formulation appropriate for pulmonary delivery
in at least one of the one or more concave indentations, wherein the
brittle matrix medicinal formulation comprises a non-tightly packed
porous flocculated web matrix comprising one or more brittle-matrix
particles of one or more active agents, wherein a portion of the one or
more brittle-matrix particles is delivered and templated by the formation
of one or more particles upon atomization from the unit-dose delivery
system using a dry powder inhaler to form a respirable porous particle
for deep lung delivery.

[0014]The present invention includes a medicinal formulation for use in a
dry powder inhaler having a non-tightly packed porous flocculated web
composition comprising one or more brittle-matrix particles of one or
more active agents, wherein a portion of the one or more brittle-matrix
particles is templated by a patient and/or device induced shearing energy
to form a porous particle for deep lung delivery

[0015]The present invention also provides method of making a dispersible
brittle templated composition for a dry powder inhaler system by cooling
a unit-dose delivery system intended for one or more metered doses for
inhalation; depositing one or more drops of a drug solution on the
unit-dose delivery system, wherein the drug solution comprises one or
more active pharmaceutical ingredients, one or more solvents, and one or
more excipients, where said drop freezes upon contact with the packaging
material; lyophilizing the pharmaceutical product to produce a
non-tightly packed brittle matrix; equilibrating the non-tightly packed
brittle matrix to room temperature; and combining the non-tightly packed
brittle matrix with a suitable dry powder inhalation device.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]For a more complete understanding of the features and advantages of
the present invention, reference is now made to the detailed description
of the invention along with the accompanying figures and in which:

[0050]FIGS. 33A-33B are images of TFF particle after lyophilization (33A)
and after drying with acetonitrile (33B);

[0051]FIG. 34A is an SEM image of BSA particles, FIG. 34B is an SEM image
of BSA:Trehalose, FIG. 34C is an SEM image of milled BSA particles, FIG.
34D is an SEM image of spray dried BSA particles, and FIG. 34E is an SEM
image of TFF particles drying with acetonitrile;

[0052]FIG. 35 is a graph of the particle sizes measured by static light
scattering for BSA spheres formed by milling and spray drying and BSA
nanorods formed by thin film freezing (TFF) suspended in acetonitrile
where closed symbols indicate sonicated powder and open circles indicate
unsonicated powder;

[0055]FIG. 38 is a graph of the particle sizes measured by static light
scattering for BSA nanorods from thin film freezing (TFF) suspended in
HFA 227 or HPFP where closed symbols indicate sonicated powder and open
circles indicate unsonicated powder;

[0056]FIG. 39A is an optical image of TFF particles after HFA 227
evaporation and FIG. 39B is an SEM image of TFF particles after
sonication and HFA 227 evaporation;

[0058]FIG. 41A is a graph of the ACI mass deposition profiles for device
(D) and spacer and throat (S+T) and stages 0-7 and FIG. 41B is a graph of
the APS mass distribution with a formulations on bar charts include BSA
(diagonal lines), BSA+Tween 20 (horizontal lines), and BSA:Trehalose
1:1+Tween 20 (dotted);

[0060]FIG. 43 is a table of the dosage and aerodynamic properties of TFF,
milled, and spray dried particle suspensions in HFA 227;

[0061]FIG. 44 is a table of the aerodynamic particle sizes determined by
ACI and APS and geometric particle sizes determined by laser diffraction
and SEM;

[0062]FIG. 45 is a table of the calculation of the van der Waals (VdW)
interaction potential Φvdw of BSA particles in HFA 227;

[0063]FIG. 46 is a table of the settling behavior of BSA particles
prepared by TFF, milling, and spray drying and calculations for porous
shell particles prepared by spray drying, with the aValue determined from
the equivalent volume of a sphere measured from laser light scattering;
bThe density difference was determined by ρf-ρL with ρp=1.5
g/cm3; cDetermined from dimensions given by Dellamary et al.; dCalculated
for primary particle with 100 nm thick shell;

[0064]FIG. 47 is an optical image of protein pMDI formulations (Lys in HFA
227 with a drug loading of 20 mg/mL, Lys in HFA 134a with a drug loading
of 40 mg/mL, 50 mg/mL, 90 mg/mL, and BSA (BSA) in HFA 227 with a drug
loading of 50 mg/mL, left to right) 4 hours after shaking;

[0070]FIGS. 53A, 53B and 53C are scanning electron microscopy images of
TFF ITZ (FIG. 53A) before and (FIG. 53B) after exposure to HFA 227 and
(FIG. 53C) SEM image of TFF ITZ after pMDI was actuated into water,
without any exposure to air;

[0071]FIG. 54 is a graph of the dynamic light scattering (DLS)
measurements of HFA-exposed TFF ITZ in water;

[0074]FIG. 57 is a graph of the aerodynamic diameters of milled, TFF, and
CP drug compositions measured by the APS 3321/3343 and the
Aerosizer/Aerodisperser systems;

[0075]FIG. 58 is a graph of the aerodynamic particle size distribution for
the TFF lys composition;

[0076]FIGS. 59A-59C are SEM micrographs of (FIG. 59A) TFF lys nanorods
prior to aerosolization and (FIG. 59B) after aerosolization and FIG. 59C
is an image at higher magnification of aerosolized TFF lys particles; and

[0077]FIG. 60 is a graph of the aerodynamic distribution of brittle-matrix
particles emitted from an ADVAIR DISKUS®.

DETAILED DESCRIPTION OF THE INVENTION

[0078]While the making and using of various embodiments of the present
invention are discussed in detail below, it should be appreciated that
the present invention provides many applicable inventive concepts that
can be embodied in a wide variety of specific contexts. The specific
embodiments discussed herein are merely illustrative of specific ways to
make and use the invention and do not delimit the scope of the invention.

[0079]The present inventors recognized that the delivery of protein
therapeutics has been largely limited to parenteral delivery due to the
chemical and physical instabilities of proteins and challenges in
permeating biological membranes. The present inventors also recognized
that pulmonary delivery is non-invasive routes offers advantages of large
alveolar surface area (about 100 m2), rapid absorption across the
thin alveolar epithelium (between about 0.1 and about 0.5 μm),
avoidance of first pass metabolism, and sufficient bioavailabilities.

[0080]For pulmonary delivery, pressurized meter dose inhalers (pMDI)
remain the most popular delivery device, relative to dry powder inhalers
(DPI) and nebulizers, because of low cost, portability, and
disposability. Because most drugs, including proteins, are insoluble in
hydrofluoroalkane (HFA) propellants, most effort has focused on the
design of stable suspensions. The lack of understanding of how to form
these stable suspensions has limited the development of viable
formulations. Although certain proteins in suspensions may potentially be
natured by HFAs, the low degree of contact in the solid state with the
solvent, relative to solutions, is highly beneficial in some instances,
e.g., insulin, lysozyme, catalase and rhDNase I.

[0081]To achieve high deposition of aerosolized particles in the deep
lung, the aerodynamic diameter (da) should range between about 1-5
mm. Such protein particles may be produced by milling, spray drying, and
spray freeze-drying (SFD). Milling processes can generate significant
amounts of heat on localized areas of the protein particle which can lead
to denaturation. In spray drying and SFD processes, proteins may adsorb
and subsequently denature and aggregate at the large gas-liquid interface
created upon atomization of droplets on the order of about 10-100 mm,
although this effect may be mitigated with interfacially active
excipients. Limited process yields, in terms of weight of protein, for
spray drying (about 50-70%) and SFD (about 80%) are a major concern for
highly valuable proteins.

[0082]The present inventors recognized that methods and devices currently
used in the art have a significant challenge in producing protein
particles with over about 90% yield, the optimal da for deep lung
delivery, and high stability against aggregation. In fact, there have
been few suspensions in the art that provide a 1-5% (w/w) mass loadings
in HFAs and are stable against settling on time scales of over 60
seconds. As the mass loading increases up to and above 5% (w/w),
particles often aggregate within aerosolized droplets leading to
substantial increases in da and thus reduction in fine particle
fraction (FPF).

[0083]Flocculation and settling can lead to irreversible particle
aggregation as well as variable dosing between actuations. For example,
suspensions of spherical particles formed by milling or spray drying
often flocculate and settle in less than 60 seconds. Consequently, the
efficiency of pMDIs is often limited for suspensions of proteins, as well
as low molecular weight drugs, with typical FPFs between about 5-30%.
Although surfactants and co-solvents, such as ethanol, could potentially
stabilize the suspension, the surfactants currently approved by the FDA
for inhalation are insoluble in HFAs. Even for soluble surfactants, the
surfactant tails are often not solvated well enough by HFAs, which have
low polarizabilities and van der Waals forces, to provide steric
stabilization. Thus, the present inventors have developed a new
surfactant structures by achieving a fundamental understanding of the
molecular interactions with atomic force microscopy and theory. The
present inventors have also developed a method to minimize the use of
co-solvents that can chemically destabilize drugs and modify protein
conformation.

[0084]An alternative approach is to modify the particle morphology to
enhance the colloidal stability of the primary particles. Large porous
particles or hollow particles with porous or nonporous shells formed by
spray drying were stable against settling for at least about 4 hours when
suspended in HFAs. Respirable fractions were as high as 68%. Here, the
presence of pores filled with HFA decreases the density difference of the
particle with the surrounding HFA media and reduces van der Waals
attractive forces between particles. Additional reports of settling
rates, primary particle aggregation, and changes in fine particle
fraction, especially after storage, will be beneficial for further
understanding this approach. Recently, large porous nanoparticle (LPNP)
aggregates, with da optimized for dry powder inhaler (DPI) pulmonary
delivery, have been formed by spray drying of aqueous suspensions of
submicron particles.

[0085]Upon contact with lung tissue, these particles break up into
nanoparticles to facilitate dissolution and absorption. To extend this
approach to delivery with a pMDI, each LPNP can be stabilized as an
individual entity in a colloidal dispersion as shown in FIG. 1, if the
LPNPs do not aggregate and settle. An alternative approach for efficient
nanoparticle delivery to the deep lung is to nebuilize nanoparticle
dispersions in aqueous media.

[0086]Spray freezing into liquids (SFL), and thin film freezing (TFF),
have been shown to produce high surface area, stable rod-like particles
with about 50-100 nm diameters and high aspect ratios, despite slower
cooling rates than in SFD. The stability of lactase dehydrogenase, based
on enzymatic activity, was increased in these processes relative to SFD.
This increase was achieved by lowering the area of the gas-liquid
interface, which has been shown to denature proteins.

[0087]The present invention provides a method of forming suspensions
against settling stable of BSA particles in HFA 227 without stabilizing
surfactants or co-solvents in order to achieve high fine particle
fractions in pMDI delivery. In stark contrast to the methods currently
used in the prior art, the present invention provides a method of
purposely flocculate the particles in the HFA to prevent settling (i.e.,
the opposite of the prior art). Spheres, produced by milling or spray
drying, were added to HFA 227, but they produced dense flocs that settled
rapidly. Asymmetric particles, such as rods, may be expected to pack less
efficiently to form much lower density flocs with greater free volume
than spheres. Rods were produced by TFF.

[0088]FIGS. 1A-1D are SEM images of URF particles from surfactant free
formulations. The present invention provides very light open flocs in an
HFA that occupy the entire vial and stack upon each other to prevent
settling for months, as illustrated in FIG. 1. The morphology was
determined by SEM of the original particles and after solvent removal of
particles suspended and sonicated in acetonitrile or HFA 227. The
flocculation is reversible, in that the flocs break up into submicron
primary rod particles upon transfer to a more polar solvent acetonitrile.
The particles were also studied in 2H,3H perfluoropentane (HPFP), a
non-volatile surrogate for HFA 227, to analyze floc size by optical
microscopy and static light scattering. The da values were
determined with an Andersen cascade impactor (ACI) and aerodynamic
particle sizer (APS) and dg values with static light scattering and
SEM micrographs. The emitted HFA droplets, on the order of about 25
μm, were utilized to break apart and template the highly open flocs as
seen in FIG. 1. Upon evaporation of the HFA, the shrinkage of the flocs
from capillary forces produces smaller and denser porous particles with
desirable da.

[0089]The particle volume fractions and fractal dimensions for flocs
composed of either cylindrical (rods) or spherical primary particles have
been characterized. Calculations of van der Waals energies between
suspended particles are presented to explain floc formation and break up
of the floc into subdomains upon templating the flocs with the HFA
droplets. The particle shrinkage during HFA evaporation leads to the
final aerosolized particle size and porosity as explained with a material
balance. The present invention provides a novel approach of flocculating,
templating, and shrinking the particles results in proper da with
low polydispersities without surfactants or co-solvents. Thus, the
present invention circumvents the classical paradigm of attempting to
stabilize colloidal dispersions of preformed primary particles with
surfactants. The flocculation for achieving stable suspensions and high
fine particles fractions without the need for surfactants of the present
invention is of practical interest for wide classes of low and high
molecular weight pharmaceuticals and biopharmaceuticals that can be
formed into nanorods.

[0090]Dry powder inhalers may use the flocs of asymmetric particles for
dose delivery. Currently dry powder inhalers do not use flocs of
asymmetric particles with high aspect ratios.

[0091]The flocs can break up more easily under the influence of the shear
forces in the dry powder inhaler than more dense particles with lower
aspect ratios. The break up of the flocs will produce smaller flocs
composed of particles with appropriate aerodynamic diameters for deep
lung delivery. Currently, the efficiency of delivery by dry powder
inhalers can be limited by the inability of the air to break up the
particles. Furthermore, small high aspect ratio primary particles that
reach the deep lung will have higher dissolution rates, as a consequence
of higher surface areas. Most of the benefits described for therapy with
flocs composed of anisotropic particles described in this application
will also be present for delivery with dry powder inhalers. The particle
may be loaded into the dry powder inhaler by a variety of methods. They
may be compacted into blister packs in the solid state. They may also be
loaded as colloidal suspensions in a solvent, where the solvent is a
liquid, compressed gas, for example a hydrofluoralkane. The evaporation
of the solvent may be used to compact the flocs to raise the final
particle density in the dry powder inhaler. In addition, the flocs may be
formed directly in a component of the dry powder inhale device by thin
film freezing. As described above for PMDIs, this approach does not use
particles that are pre-formed to design the aerodynamic diameter of the
aerosol particle. Instead, the aerodynamic diameter is generated in the
air ways by the shear forces upon rupture of the flocs. This aerodynamic
diameter is not present in the starting flocs. Thus, the present
invention circumvents the classical paradigm of attempting to design the
aerodynamic diameters of pre-formed individual particles prior to loading
into the dpi.

[0093]BSA powders were prepared by the thin film freezing (TFF) process
described previously. Briefly, 5 mg/mL feed solution of BSA in 10 mM
pH=7.4 potassium phosphate buffer was passed at a flow rate of 4 mL/min
through a 17 gauge (e.g., 1.1 mm ID, 1.5 mm OD) stainless steel syringe
needle. The droplets fell from a height of 10 cm above a rotating
stainless steel drum (12 rpm) 17 cm long and 12 cm in diameter. The
hollow stainless steel drum was filled with dry ice to maintain a drum
surface temperature of 223 K. On impact, the droplets deformed into thin
films and froze. The frozen thin films were removed from the drum by a
stainless steel blade and transferred to a 400 mL PYREX® beaker
filled with liquid nitrogen. The excess liquid nitrogen was evaporated in
a -80° C. freezer.

[0094]A Virtis Advantage Lyophilizer (The Virtis Company, Inc., Gardiner,
N.Y.) was used to dry the frozen slurries. Primary drying was carried out
at -40° C. for 36 hrs at 300 mTorr and secondary drying at
25° C. for 24 hrs at 100 mTorr. A 12 hour linear ramp of the shelf
temperature from -40° C. to +25° C. was used at 100 mTorr.

[0095]Spray drying was performed with a Buchi Model 190 mini spray dryer
(Brinkmann, Westbury, N.Y.). A 10 mg/mL BSA feed solution in 10 mM
potassium phosphate buffer (pH=7.4) was atomized using a 0.5 mm ID two
fluid nozzle with an atomizing air flow rate of 200 mL/s. The liquid
protein formulation was pumped through the nozzle by a peristaltic pump
(VWR, Bridgeport, N.J.) at a flow rate of 5 mL/min using 5 mm ID silicone
tubing. The inlet temperature for the heated aspirator air was set to
150° C. at a flow rate of 1000 L/hr. The resulting outlet
temperature from the above conditions was 80° C.

[0096]Bulk BSA powder as received was suspended at 5 mg/mL in
acetonitrile. The BSA suspension was placed in a mill filled with 50
ceramic balls approximately 1 cm in diameter and milled on a mechanical
roller for 24 hours. The milled BSA suspension was dried in the Virtis
Advantage Lyophilizer at a shelf temperature of 30° C. for 12
hours at 1000 mTorr.

[0097]Dry powders were placed in 60 mL glass bottles (Qorpak, Bridgeville,
Pa.) and pre-cooled in a -80° C. freezer. HFA 227 was also
pre-cooled in a -80° C. freezer and poured into the bottles
containing the protein powders to form 0.7% (w/w) suspensions. The
bottles were packed in dry ice and the suspensions were then sonicated
for 2 minutes using a Branson Sonifier 450 (Branson Ultrasonics
Corporation, Danbury, Conn.) with a 102 converter and tip operated in
pulse mode at 35 watts. Approximately 5 mL aliquots of the suspension
were then dispensed into a 500 mL acetonitrile bath for particle size
analysis by static light scattering with a Malvern Mastersizer-S (Malvern
Instruments, Ltd., Worcestershire, UK). Typical obscuration values ranged
from about 11 to about 13%. Next, 10 mL of the cooled protein
formulations were dispensed into 17 mL glass pMDI aerosol vials (SGD,
Paris, France) and fitted with metering valves containing 100 μL
metering chambers (DF10 RC 150, Valois of America, Inc., Congers, N.Y.).
The vials were then allowed to warm up to room temperature.

[0098]The dried powders were also suspended in acetonitrile at a
concentration of 5 mg/mL and sonicated for about 2-3 minutes in the same
manner described above. Approximately 2 mL of the sonicated suspension
was dispersed into a 500 mL acetonitrile bath and the particle sizes were
analyzed by static light scattering.

[0099]The amount of BSA was measured using the Micro BCA Protein Assay
following protocols provided by Pierce (Rockford, Ill.). Each sample was
measured in triplicate with relative standard deviations (% RSD) <2%.
The absorbance of the solutions was measured at 562 nm in a 96 well plate
spectrophotometer (μQuant Model MQX200; Biotek Instruments Inc.,
Winooski, Vt.). Untreated BSA was used to prepare the protein standards
at concentrations between about 2 and 30 μg/mL.

[0100]The protein suspensions in HFA were actuated once through the firing
adaptor of a dosage unit sample tube (26.6 mm ID×37.7 mm
OD×103.2 mm length; 50 mL volume; Jade Corporation, Huntingdon,
Pa.). The firing adaptor was removed, and 40 mL of DI water was added to
dissolve the protein. The sampling tube was shaken and allowed to sit for
at least 30 min. to assure that the protein was dissolved in water. The
protein concentration was determined using the Micro BCA protein assay in
conjunction with the μQuant spectrophotometer. The glass vial
containing the HFA protein suspension was weighed before and after each
actuation to assure that the proper dose had been released. The
measurement was repeated 3 times to get an average dose delivered through
the valve (DDV) for each formulation.

[0101]To characterize the aerodynamic properties of the particles, an
eight-stage Andersen cascade impactor (ACI) (Thermo-Andersen, Smyrna,
Ga.) with an attached 15 cm spacer and an air flow-rate of about 28.3
L/min was used to quantify mass median aerodynamic diameter (MMAD),
geometric standard deviation (GSD), fine particle fraction (FPF), and
emitted dose (ED). Initially 3 shots were sent to waste, and the next 5
shots were made into the ACI. The interval between shots was between
about 15-30 seconds to prevent cooling of the metering chamber and
subsequent moisture condensation. After the last dose was discharged, the
glass vial was removed from the impactor and the valve stem and actuator
were rinsed separately with a known volume of DI water. Each plate of the
impactor was placed in a separate container with a known volume of DI
water and soaked for 30 minutes to assure complete dissolution. The
protein concentrations were then measured with the Micro BCA Protein
Assay.

[0102]The da of the protein particles were also determined in
triplicate with an Aerodynamic Particle Sizer (APS) 3321 (TSI, Shoreview,
Minn.). The throat and spacer from the ACI were placed over the inlet of
the APS and the airflow rate through the inlet was 5 L/min. Each
formulation was shot once through the spacer and throat. The particle
size range by mass was determined with the Aerosol Instrument Manager
(AIM) software provided by TSI.

[0103]To obtain aerosolized particles for scanning electron microscopy
(SEM) (Hitachi Model S-4500, Hitachi Ltd, Tokyo, Japan) analysis, double
carbon adhesive tape was applied to stage 3 of the ACI. Each formulation
was actuated once through the ACI with an air flow rate of 28.3 L/min.
The carbon tape was removed from stage 3 and applied to an aluminum SEM
stage, which was transferred rapidly to a Pelco Model 3 sputter-coater to
minimize exposure to moisture. Total exposure to the atmosphere was less
than 1 minute. The SEM micrographs were then characterized with imaging
software (Scion, Frederick, Md.) to determine the particle size
distribution of at least 100 particles.

[0104]The aerosolized particles were also characterized by static light
scattering. Each formulation was actuated once through the ACI spacer and
throat. The aerosol exited the outlet of the throat downwards 5 cm
directly above the laser beam of the Malvern Mastersizer S. For each
formulation 100 measurements of the aerosolized spray were made every 5
ms. The recorded measurements were then averaged to give the final
profile of the aerosolized particles on a volume basis.

[0105]Moisture contents in the vials of each formulation were tested with
an Aquatest 8 Karl-Fischer Titrator (Photovolt Instruments, Indianapolis,
Ind.) according to the method described by Kim et al. A 19 gauge needle
was inserted through the septum of the titration cell with the needle tip
placed below the reagent, and each formulation was measured in
triplicate. For all formulations tested the moisture content was
approximately 500 ppm. The pure HFA was found to have a moisture content
of 250 ppm. The total amount of moisture to the amount of protein
particles was 7% (w/w).

[0106]The particles were initially dispersed by pipette mixing in HPFP and
were observed for about 2 minutes with a Nikon OPTIPHOT 2-POL optical
microscope with an attached MTI CCD-72× camera (Nikon, Tokyo,
Japan). Pictures were taken 30 and 60 seconds after initial dispersion in
HPFP.

[0107]FIG. 2 is a table of URF ITZ powder dispersed in HPFP. The μQuant
spectrophotometer was used to measure turbidity at 350 nm to characterize
BSA aggregation. Dry powders of BSA were reconstituted to 1 mg/mL and
3×300 uL aliquots of each formulation were placed in a 96 well
Falcon plate which was set in the spectrophotometer.

[0108]Particles of BSA suspended in acetonitrile were analyzed by a
custom-built dynamic light scattering (DLS) apparatus. The scattering
angle was set to 90° and the data were analyzed a digital
autocorrelator (Brookhaven BI-9000AT) and a non-negative least-squares
(NNLS) routine (Brookhaven 9KDLSW32). The suspension concentration was
0.5 mg/mL which gave a measured count rate of approximately 150 kcps.
Measurements were made over a period of about 2 minutes.

[0109]Approximately 100-300 mg of protein powder was loaded into a 100 mL
graduated cylinder. The tap density of the protein particles was measured
with a Vankel tap density meter (Varian, Palo Alto, Calif.).

[0110]FIGS. 3A and 3B are SEM images of pMDI formulation, while 3C and 3D
are the corresponding graphs of particle size. The fluffy BSA particles
made by TFF shown in FIG. 2A had a low tap density of 0.0064 g/cm3.
The morphology of the BSA powder prepared by TFF was interconnected rods
50 nm in diameter as seen in FIG. 3A. With the addition of 5 mg/mL
trehalose to the BSA feed solution, similar rods were produced, as well
as fine 50-100 nm relatively spherical particles FIG. 3B. Similar
morphologies were observed previously for lysozyme produced by TFF at 223
K. The BSA particles prepared by wet milling as seen in FIG. 3C did not
have high external porosity like the TFF particles, but were in the form
of cubes with smooth sides with 400-800 nm dimensions. Lastly, spray
drying BSA at a feed concentration of 10 mg/mL formed protein particle
spheres 3-6 μm in diameter with smooth surfaces as seen in FIG. 3D.

[0111]For characterization by static light scattering, the various BSA
particles suspended in acetonitrile were sonicated for about 2 minutes.
FIGS. 4A-4B are TEM images of URF ITZ aerosol from pMDI. As shown in FIG.
4 the d(v,50) values were 330 nm, 410 nm and 6.3 μm for the TFF,
milled and spray dried BSA particles, respectively, consistent with the
sizes in the SEMs. Thus, the primary particles remain dispersed in
acetonitrile and do not aggregate. As demonstrated previously with
lysozyme, the cooling rate in the TFF process for BSA was sufficiently
fast to form high surface area powders that redisperse to 330 nm
particles in acetonitrile, with little sonication (less than about 2
minutes). As a further indication of high tendency of the nanorods to
deaggregate and disperse in acetonitrile, even with no sonication 2 peaks
were observed with maxima at 330 nm and 20 μm, with approximately 50%
of the particles by volume below 1 μm as shown in FIG. 4. Thus the
aggregation of the nanorods in the powder state is highly reversible.

[0112]To compliment the light scattering results by SEM, the sonicated
suspensions in ACN were frozen by drip freezing into liquid nitrogen. The
acetonitrile was then removed by lyophilization leaving fluffy particles
with an approximate tap density of 0.012 g/cm3 (FIG. 2B). When the
particles were redispersed in acetonitrile the measured particle size
profile was d(v,50)=330 nm which was similar to the profile in FIG. 4 of
the original TFF dispersion, indicating that the lyophilization process
did not cause irreversible particle aggregation. As observed by SEM, the
morphology in FIG. 3E were 50-100 nm diameter rods, similar to the
interconnected rods of the original TFF powder in FIG. 3A, and consistent
with the sizes from light scattering results in FIG. 4. Thus exposure to
acetonitrile followed by sonication does not alter the morphology
significantly.

[0113]FIGS. 5A and 5C are data for the 100% Itz URF samples shown in the
SEM images FIGS. 5B and 5D. FIG. 5E is a XRD of URF ITZ powder. The dried
TFF BSA particles were suspended in HFA 227 and acetonitrile (ACN) at
0.70% (w/w) corresponding to a volume fraction in the vial φv of
0.0077, as determined from the true density of BSA ρp=1.3
g/cm3 as shown in FIG. 5. As shown in FIG. 5A, the particles did not
settle even after 1 year in storage in HFA 227. Immediately upon adding
HFA, the particles formed flocs that filled the entire volume of the
vial. For a control with an extremely low φv of only 0.070%
(w/w) as shown in FIG. 5B the loose buoyant flocs still filled
approximately half the HFA volume. For the milled BSA nanoparticles, the
suspension initially appeared to be uniform (as in FIG. 5A), but the
particles settled to the bottom after only 5 minutes as shown in FIG. 5C.
Since these particles settled in HFA 227 (1.41 g/cm3), the milling
may have compacted the particles to ρ above 1.3 g/cm3. These
particles creamed in HPFP (1.59 g/cm3). Thus, it was estimated that
ρp˜1.50 g/cm3, the average of the two solvent
densities. The spray dried particles dispersed well with shaking, but
creamed after only 2 minutes as shown in FIG. 5D. The TFF nanorods
suspended in acetonitrile and sonicated for 2 minutes formed a milky
uniform dispersion as shown in FIG. 5E. After 3 days the particles had
settled as shown in FIG. 5F. The dispersion/settling behavior shown in
FIGS. 5E and 5F was also observed for milled and spray dried particles in
acetonitrile (data not shown) with settling in about 3 days and about 30
minutes, respectively.

[0114]Because the vapor pressure of HFA 227 is above ambient at 25°
C. (about 500 kPa), the particles were not studied in situ by microscopy
or light scattering. Instead, the particles were studied at ambient
pressure in HPFP, a surrogate nonvolatile solvent. Because HPFP has a
similar polarity and polarizability as HFA 227, attractive forces between
solutes such as budesonide are similar in both solvents on the basis of
atomic force microscopy (AFM). FIG. 6 is a dissolution graph of particles
emitted by pMDI. According to light microscopy (FIG. 6A), the TFF
particles in HPFP were in the form of loosely packed aggregates of rods
as shown in FIG. 6B and FIG. 6C). The particles were in 200-300 μm
flocs with subdomains on the order of 25 μm within 5 seconds after
dispersing the particles by pipette mixing (see FIGS. 6A and 6B). For the
spray dried (as shown in FIGS. 6D and 6E) and milled (as shown in FIG.
6F) particles, 100 μm flocs formed in 30 seconds and grew to over 200
μm in 60 seconds.

[0115]FIGS. 7A-7B are SEM images of Charleston sample Dow amorphous ITZ.
These flocs were more densely packed and composed of larger primary
particles than those formed from TFF particles. These sizes were
consistent with static light scattering measurements of the sonicated and
unsonicated suspensions in HPFP with d(v,50) values between about 215-259
μm.

[0116]To better anticipate the fate of particles throughout the pMDI
delivery process, it would be beneficial to determine how reversibly the
nanorods are bound together in the flocs. The elevated pressure of the
HFA complicates in situ light scattering. Furthermore, the HFA suspension
could not be lyophilized to prepare a sample for SEM since the freezing
point (-131° C.) of HFA 227 is too low to for conventional shelf
lyophilizers. To investigate the effect of HFA evaporation on the
particles, HFA was cooled to -80° C., well below the boiling point
of -16° C., and completely evaporated. The TFF particle residue
only occupied approx. 1 mL (tap density of 0.10 g/cm3, FIG. 8A), an
order of magnitude less than that of the starting TFF bulk powder as
shown in FIG. 2A.

[0117]FIGS. 8A-8C are SEM images of Charleston sample Dow amorphous ITZ
from pMDI. The morphology shown in FIG. 8A was rods with 100 nm diameters
(see FIG. 8B), similar to the original TFF particles in FIG. 3A.
Therefore, exposure to HFA 227 followed by sonication did not
significantly alter the microscopic nanorod morphology. However, the
densified aggregates of the nanorods formed by capillary forces upon
evaporation as shown in FIG. 5B of HFA were not redispersible in HFA or
in ACN. For a sonicated TFF paticle dispersion in ACN, the lyophilized
powder was redispersible in ACN and in HFA, forming suspensions identical
to FIG. 5A. Thus it appeared that the capillary forces during HFA
evaporation and perhaps moisture produced irreversible aggration of the
nanorods.

[0118]Given the challenges of in situ high pressure light scattering,
lyophilization of HFA 227, and compaction of the TFF rods by capillary
forces upon HFA evaporation, a more practical approach was to transfer
the suspension from HFA 227 to a less volatile solvent. If the nanorods
redisperse to primary particles in a good solvent such as acetonitrile,
then they were not aggregated irreversibly in HFA 227. A 2 mL aliquot of
the cold TFF suspension was mixed directly with 500 mL of acetonitrile at
25° C. The flocs deaggregated nearly completely to individual
primary particles with over 80% of the volume distribution between 100 nm
and 1 μm, and a maximum at 11 μm as shown in FIG. 7. A relatively
small peak was centered at 5 μm. The distributions nearly matched
those of the original TFF particles in ACN. In a complimentary
experiment, the valve of the pMDI containing was submerged into
acetonitrile and actuated. A slightly turbid dispersion was formed with
an approximate particle concentration of 0.5 mg/mL, too low for detection
by static light scattering, but not for DLS.

[0119]FIGS. 9A-9B are graphs and 9C is a SEM characterizing the ITZ sample
made by CP. From DLS, the particle size was 1-2 μm much smaller than
the 250 μm floc size in HFA. Therefore, both experiments indicate the
loosely connected flocculated nanorods in HFA were reversible and broke
up into primary nanorods, which will be shown to be beneficial for lung
delivery.

[0120]Aggregates of protein molecules did not appear to form according to
optical density (OD) measurements at 350 nm of 1 mg/mL BSA [43,61]. The
OD was the same at 0.042 for aqueous solutions in 10 mM phosphate pH=7.4
buffer prepared from bulk and TFF powder, both before and after storage
in HFA 227 for 1 week. In the glassy state, BSA is less susceptible to
aggregation. The total moisture to BSA content was 7% (w/w) for the
suspended BSA particles in HFA 227 as determined by Karl-Fischer
titration. Even at particle moisture contents of 8% (w/w), BSA glass
transition temperatures Tg range between 80-100° C. Thus the
temperature was well below Tg, assuming the HFA 227 did not
contribute to plasticization.

[0121]The suspension must be stable for consistent dosing with a pMDI,
which is commonly characterized by the dose (mass) delivered through the
valve (DDV) as seen in Table 1.

[0122]The concentration was 10 mg/mL or 0.7% (w/w) in each HFA suspension.
Therefore, the theoretically delivered dose per actuation would be 1 mg
with the 100 μL valve. For the BSA TFF particles, the DDV values were
92% and 63% of the theoretical delivery dose for the sonicated and
unsonicated TFF particles, respectively as seen in TABLE 1. For the
BSA:Trehalose 1:1 formulation, it was 90%, and the delivered dose was 450
μg/actuation as a consequence of the lower amount of BSA loaded into
the vial. For the milled and spray dried suspensions with rapid settling,
the DDV was only 30-31% of the theoretical loading. Here, the formulation
was actuated less than 5 seconds after vigorous shaking Therefore, these
suspensions were not tested further for aerosol properties.

[0123]FIGS. 10A-10C are SEM images of ITZ made by CP from pMDI. As shown
in Table 2 and FIG. 10, the da determined from the Andersen cascade
impactor (ACI) and the Aerodynamic Particle Sizer (APS) were in good
agreement and ranged from 3 to 4 μm, within the optimal 1-5 μm
range for pulmonary delivery.

[0124]As determined by the ACI, the fine particle fraction (FPF)
(particles less than 4.7 μm) was unusually high [32] for an HFA
suspension, ranging from 38 to 48%, compared to 5 to 30% for typical
suspensions [32], producing a fine particle dose/actuation of
approximately 300 μg for the first two formulations in TABLE 1. The
emitted dose (ED) (amount of drug that exited the actuator) was
approximately 70% of the DDV upon actuation (see TABLE 1 and FIG. 10A).
The addition of Tween 20 did not affect any of the properties of the
aerosolized TFF powders in TABLE 1 significantly or the suspension
stability, indicating that it was not needed as a stabilizer.

[0125]The particles were recovered from the ACI for SEM analysis. The peak
drug mass in the ACI was deposited on stages 3 and 4, with da
between 2.0-4.7 μm as shown in FIG. 10A. Therefore, particles were
collected on stage 3 (da=3-4 μm).

[0126]FIG. 11 is a table comparing ITZ formulations. The particles were
porous and composed of rods with diameters less than 500 nm (see FIGS.
11A and 11B), similar in morphology to the original nanorods in FIG. 3A.
For BSA:Trehalose 1:1 the fine 50-100 nm primary particles, shown in FIG.
3B, changed morphology to include curved plates with features on the
order of more than one micron as shown in FIGS. 11C and 11D.

[0127]The SEMs were analyzed by Scion software o determine the volume
average diameter

D Vol = d 4 d 3 ( 1 ) ##EQU00001##

where d is the measured diameter of the particle. The Dvol for BSA
was approximately 9 μm, while for BSA:Trehalose 1:1 it was slightly
smaller, 7 μm (TABLE 2). The dg of the aerosolized particles were
also measured by static light scattering. An effective refractive index
ne was calculated according to the Bruggeman mixing rule [66] based
on the volume fraction of BSA in the aerosolized particle φg.
From the dg and the da (see Table 2), the particle density
ρg can be defined by

da=dg {square root over (ρg)} (2)

where ρg=0.19 g/cm3. The resulting
φg=ρg/ρp=0.14. With n=1.45 and 1.00 for pure
BSA and air, respectively, ne=1.1. As shown in TABLE 2 the volume
average d(v,50) particle sizes varied by less than 1 μm from the
values determined from the SEM micrographs. The consistent dg and
da, each measured by two techniques, indicate that TFF particles
form large porous particles, and with the optimal size range for
pulmonary delivery upon aerosolization. When the TFF particles were
actuated above 10 mM phosphate buffer (pH=7.4) the porous particles were
observed to dissolve in less than 5 seconds. The high surface area favors
rapid dissolution, which could be advantageous for rapid dissolution
rates of proteins that have low solubilities in water.

[0128]The van der Waals forces between particles play a key role in the
differences in colloidal stabilities of various types of primary
particles and the behavior of the flocs in this study, as depicted in the
summary in FIG. 1. According to the Derjaguin-Landau-Verwey-Overbeek
(DLVO) theory, particle stability depends on counteracting the attractive
van der Waals forces by electrostatic and/or steric repulsion. If
attractive van der Waals (VdW) forces are dominant at all separation
distances, particles flocculate and may then settle. Currently,
electrostatic stabilization in HFAs is not well understood, but atomic
force microscopy (AFM) measurements indicate that electrostatic forces
may be negligible compared to attractive VdW forces. The understanding of
steric stabilization in HFAs is in its infancy. While novel surfactants
are being discovered, developed and approved, alternative mechanisms form
the formation of stable suspensions in HFAs without surfactants would be
useful.

[0129]The destabilizing van der Waals attractive forces between suspended
are weaker for porous particles or hollow particles with thin solid
shells. These particles can be stable for hours in HFAs, compared to
non-porous 1-5 micron particles, which often flocculate and settle
rapidly in less than 1 minute (see TABLE 2). Dellamary et al. suggested
that the increased suspension stability resulted from a weaker attractive
VdW energy potential Φvdw between the particles (FIG. 1A), but
quantitative calculations were not presented.

[0130]As shown in the Appendix the van der Waals energy Φvdw is
directly proportional to the Hamaker constant A121. In order to
compare values of Φvdw it is necessary to choose a separation
distance, D, between particles. TABLE 3 gives the D where Φvdw
becomes equivalent to the thermal energy 3/2 kBT at 298K.

[0131]An increase in D required to overcome thermal energy indicates
stronger attraction between particles. In TABLE 3, the porous particles
with φ=0.5 had a calculated A121 (Eq. A.3) that was nearly a
factor of 4 lower than for the non-porous particles. Consequently, D was
a factor of 3 smaller. The hollow spheres from TEM images were estimated
to have 2-5 μm diameters and about 100 nm thick shells. Although the
A121 for the hollow sphere particles with solid shells was the same
as for the non-porous particles, the calculated D was still lower by a
factor of 2 as a consequence of the differences in the geometries (Eq.
A.5). Therefore, the Φvdw calculations quantify the benefits of
weaker attraction for porous particles or for particles with hollow
cores. A reduction in Φvdw or in D to overcome thermal energy
can reduce the rate of flocculation over orders of magnitude as described
by the stability ratio.

[0132]Although, the porous or hollow sphere particles can effectively
prevent flocculation, the particles are still subject to settling by
gravity. If porous or hollow sphere BSA particles were suspended at
φv=0.0077, the particles would occupy about 10% of the
suspension (as shown in

[0133]FIG. 1A) and could potentially settle into a dense sediment. As
shown in TABLE 4, the calculated settling rate for a single hollow sphere
particle with a solid shell is 6.4×10-4 mm/s indicating that
the particles would settle a distance of 2 cm in about 9 hours. The
settled particles would then potentially aggregate irreversibly leading
to decreased FPFs upon aerosolization.

[0134]The concept in this study of stabilizing suspensions with purposely
flocculated rods is based on the space filling properties of the rods and
the flocs. Experimental and theoretical studies indicate that rods create
extremely low density flocs and thus fill much greater space compared to
spheres as illustrated in FIGS. 1B and 1C. For spheres, the volume
fraction of primary particles within a floc φf is related to the
floc diameter dfloc, primary particle diameter dp, and fractal
dimension Df, which characterizes the floc structure, by

φ f ≈ ( d floc d p ) D f - 3 ( 3 )
##EQU00002##

[0135]Philipse et al. modified Eq. 3 to account for the packing physics of
cylindrical rods of length L and diameter D with the result

φ f ≈ 1 r ( d floc V p 1 / 3 ) D f - 3
( 4 ) ##EQU00003##

where r=L/D is the aspect ratio. The volume of a TFF cylindrical rod,
Vp=0.019 μm3, was calculated from the equivalent volume of a
sphere with particle diameter d(v,50)=0.33 μm, which was measured by
static light scattering (as shown in FIG. 4A) in acetonitrile. For a rod
with volume Vp=πD2L/4 and D=0.050 μm (as shown in FIG.
3A), L is determined as 0.48 μm and thus r=9.6. For r˜10, the
predicted φf in Eq. 4 is ˜1 order of magnitude lower than
for spherical particles with equivalent dfloc, Df, and where
dp for spheres scales as Vp1/3 for rods.

[0136]The density of a floc ρf and φf can be determined
experimentally from the visually observed floc settling rate, Uf ,
according to Stoke's law

U f = d floc 2 ( ρ f - ρ L ) g 18 μ
( 5 ) ##EQU00004##

[0137]where ρL and μ are the liquid density and viscosity,
respectively, and dfloc=250 μm for TFF flocs and 100 μm for
spray dried and milled flocs. After solving for ρf in Eq. 5,
φf may be determined by the straightforward material balance
ρf=ρL+φf(ρp-ρL). As seen in
Table 4, φf for the TFF particles is 1-2 orders of magnitude
lower than for the spherical milled and spray dried particles. From Eq. 3
and 4 the calculated Df values are in a narrow range from 2.4 to 2.6
in each case. Although the milled and TFF particles have nearly
equivalent dfloc and Df values relative to the rods (as seen in
Table 4), the 1/r scaling in Eq. 5 for rods accounts for the 1 order of
magnitude decrease in φf, for a given Vp, which is
consistent with theoretical prediction above.

[0139]The extremely low φf means the flocs will fill a huge
volume of space for a given φv (as shown in FIG. 1C). The open
nanorod flocs with low φf filled large amounts of space in HFA
and stacked upon each other like tumbleweeds to prevent settling. The
volume fraction of flocs in the HFA suspension, φflocs, is given
by φflocs=φv/φf (derivation given in Appendix)
where φflocs s determines the space filling capability of the
flocs. As φflocs approaches 1 the flocs occupy the entire volume
of HFA (as shown in FIG. 1C). For the dilute φv=0.00077
suspension (as shown in FIG. 5B), the calculated φflocs was
about 0.38 (see Table 4) in good agreement with FIG. 5B. At a loading 10
fold higher, φv=0.0077, the entire vial was white without the
appearance of spaces between flocs (as shown in FIG. 5A), as expected
from the low φf . Here it was not possible to observe a settling
rate as the visual appearance did not change for 1 year, the maximum time
tested, as the φflocs of essentially unity prevented settling.
In order for the spherical particles to produce φflocs=1 the
required mass loadings for the milled and spray dried particles would be
6.7% (w/w) and 33% (w/w), respectively, compared to <0.7% (w/w) for
the TFF rods.

[0140]In contrast to the TFF rods, the hollow sphere particles would
settle the length of the vial (about 2 cm) by gravity in about 9 hours
according to Stoke's law for a particle diameter of 5 μm and shell
thickness of 100 μm. In the settled state with a high particle volume
fraction and contact between protein chains they are more likely to form
irreversible particle aggregates by interparticle diffusion and
sintering.

[0141]The open flocs in HFA 227, that gave the stable suspensions, may be
shown to be favored by the relatively strong attractive forces between
the primary particles. At first, this may seem counterintuitive to the
normal goal of lowering attractive forces to stabilize colloidal
dispersions. Upon addition of the HFA, the relatively strong attractive
forces between the primary rods, Φvdw, cause sticky collisions
to "lock in" the open structure rapidly to inhibit collapse of the flocs.
For weaker attractive forces between primary particles, collapse has been
shown to be more prevalent as particles sample a greater number of
energetically favorable locations to reduce the interfacial surface area.
Therefore, rapid flocculation from sticky collisions facilitates the
formation of low density flocs that fill the entire vial and prevent
settling.

[0142]In contrast to the flocs in HFA 227, colloidal dispersions of
primary TFF rods in acetonitrile settled in 3 days (as shown in FIG. 5F).
This settling rate agreed with the predicted settling rate of individual
effective spheres with a diameter of 330 nm from light scattering given
in Table 4. From Table 3, the calculated A121 values for BSA in
acetonitrile are 1 order of magnitude lower than in HFA 227. Therefore,
the stronger attractive forces between particles in HFA relative to ACN,
favors formation of open flocs, resulting in more stable suspensions
against settling.

[0143]Although the 250 μm flocs form stable suspensions, they are too
large to produce optimal da. The shear forces in the actuator are
needed to break apart the flocs. The calculation of these shear forces is
rarely reported because the turbulence from the immediate onset of HFA
evaporation produces complex cavitation events. According to empirical
models, aerosolized HFA droplets are typically about 10-30 μm in
diameter. Thus we choose an HFA droplet diameter of 25 μm. The shear
forces acting on the flocs are sufficiently strong to overcome the
attractive van der Waals interactions between primary particles within a
floc such that the HFA droplets may template the 250 μm flocs into 25
μm subdomains with the same φf=0.0020 as illustrated
schematically in FIG. 1. From the high φflocs (TABLE 1C) it is
expected that most of the HFA droplets are likely to be filled with a
subdomain.

[0144]Since direct comparison of calculated shear forces to van der Waals
forces of primary particles within a floc is unfeasible, the concept of
templating of the 25 μm subdomains is instead supported by a material
balance on the protein between the volume of the HFA droplet, VIIFA,
and the volume of the dry aerosolized particle, Vg, (as shown in
FIG. 1C) given by

Vgρg=VHFAρHFA (6)

where BSA concentrations are given by ρHFA=φv
ρp, and ρg=φgρp. It is assumed that
the volume fraction of particles in HFA droplet is approximately equal to
φv as a result of the break up of the flocs. From the dg
and da in Table 2 and ρg=0.19 g/cm3 (Eq. 2),
φg=0.14. The φg is nearly 20 times greater than
φv in the vial. Therefore, the capillary forces in the shrinking
HFA droplets during evaporation collapse the flocs. Eq. 6 is refined to
relate φg to φv as

φ g d g 3 = f BSA f HFA φ v d HFA 3 (
7 ) ##EQU00005##

where d is a diameter, fBSA=0.7 accounts for the mass fraction of
drug that is emitted from the actuator, and fHFA=0.5 accounts for
the mass fraction of HFA that exits the actuator orifice to form
aerosolized liquid droplets (relative to vapor).

[0145]From Eq. 7 with dg=9.3 μm (TABLE 2), dHFA=25 μm, and
φv=0.0077, the calculated φg=0.21, which compares
reasonably well to the experimentally determined φg=0.14. Also
the polydispersity in the aerodynamic properties was small. It would be
unlikely that any other factor besides templating of the flocs with
relatively uniform HFA droplets could explain these low polydispersities.

[0146]The control in FIG. 8A supports this argument since the TFF
particles remained below the meniscus of the evaporating HFA 227. The tap
density of the particles was approximately 0.10 g/cm3 (FIG. 8A)
which is within a factor of 2 of the calculated density (0.19 g/cm3)
of the aerosolized particle. Therefore, the capillary forces acting on
the TFF particles during HFA evaporation compacted the particles into
denser aggregates with a highly desirable value of the da. If
needed, the da may be manipulated further by varying the valve
volume and geometry and the HFA droplet generation. If the particles had
not collapsed partially, they would have been too large and light for
pulmonary delivery. Even after this collapse, the porosity and surface
area were still relatively high and favorable for high dissolution rates
of small molecules and proteins with limited solubilities, relative to
nonporous particles.

[0147]High (e.g., about 38-48%) fine particle fractions in HFA 227 pMDI
delivery were achieved with flocculated BSA nanorods stable against
settling for up to 1 year, without the use of surfactants and cosolvents.
Analysis of experimental settling rates of dilute suspensions indicated
that the volume fraction, φf, of the nanorods in the flocs was
an order of magnitude lower than for flocs of spherical particles
produced by milling or spray drying. The rapid and sticky attractive
collisions of nanorods, facilitates the formation of low density flocs
(250 μm) which stack upon each other to fill the entire solvent volume
to prevent settling. In contrast, denser flocs of spherical particles
filled much less space and rapidly settled within 60 seconds. The novel
concept of purposely flocculating nanorods to prevent settling is
fundamentally opposite the conventional approach of stabilizing colloidal
dispersions of primary particles. The reversibility of the nanorod flocs
in HFA 227 was demonstrated by break up of the flocs into individual 330
nm primary rod particles upon transfer to the more polar solvent
acetonitrile.

[0148]A material balance on a shrinking HFA droplet containing a 25 μm
floc subdomain predicts a final volume fraction of BSA in the aerosolized
particle in agreement with experiment. Therefore, the attractive van der
Waals interactions between primary particles within the floc are
sufficiently weak such that the atomized HFA droplets initially template
the 250 μm flocs into 25 μm subdomains. The aerosolized particles
with a da of 3-4 μm and dg of about 10 μm are optimal for
high fine particle fractions via a pMDI. The concept of forming open
flocs composed of nanorods, that are stable against settling without
surfactants, and templating the flocs to achieve optimal das and
high FPFs is of practical interest for wide classes of low and high
molecular weight pharmaceuticals and biopharmaceuticals.

[0150]FIG. 22 is a table comparing ITZ formulations. FIG. 23 is a table
comparing aerosolized particle dimensions of ACI. FIG. 24 is a graph of
the HFA droplet diameter. FIG. 25 is an illustration of the calculation
of Df. FIG. 26 is an illustration of the calculation of the settling
velocities of flocs. FIG. 27 is a SEM image of TFF particles in HPFP.
FIG. 28 is a SEM image of CP ITZ particles in HPFP. FIG. 29 is a SEM
image of DOW amorphous in HPFP. FIG. 30 is a SEM image of milled ITZ in
HPFP. FIG. 31 is a SEM image of milled ITZ in HPFP. FIGS. 32A-32C are
schematics of particle suspension of hollow sphere particles (FIG. 32A),
milled or sprayed particles (FIG. 32B) and TFF rod particles (FIG. 32C).

[0151]FIGS. 33A-33B are images of TFF particle after lyophilization (33A)
and after drying with acetonitrile (FIG. 33B). FIG. 34A is an SEM image
of BSA particles, FIG. 34B is an SEM image of BSA:Trehalose, FIG. 34C is
an SEM image of milled BSA particles, FIG. 34D is an SEM image of spray
dried BSA particles, and FIG. 34E is an SEM image of TFF particles drying
with acetonitrile. FIG. 35 is a graph of the particle sizes measured by
static light scattering for BSA spheres formed by milling and spray
drying and BSA nanorods formed by thin film freezing (TFF) suspended in
acetonitrile where closed symbols indicate sonicated powder and open
circles indicate unsonicated powder. FIG. 36A-36F are images of
suspensions in HFA 227 of TFF particles at φv=0.0077 (FIG. 36A),
φv=0.00077 (FIG. 36B), milled particles 5 minutes after shaking
(FIG. 36C) and spray dried particles at 2 minutes after shaking (FIG.
36D) at φv=0.0077, TFF particles in acetonitrile at
φv=0.0077 immediately after shaking (FIG. 36E) and 3 days after
shaking (FIG. 36F).

[0153]FIG. 42A-42D are SEM images of BSA aerosol collected from stage 3 of
Andersen cascade impactor for BSA (FIG. 42A and 42B) and BSA:Trehalose
1:1 (FIGS. 42C and 42D). FIG. 43 is a table of the dosage and aerodynamic
properties of TFF, milled, and spray dried particle suspensions in HFA
227. FIG. 44 is a table of the aerodynamic particle sizes determined by
ACI and APS and geometric particle sizes determined by laser diffraction
and SEM. FIG. 45 is a table of the calculation of the van der Waals (VdW)
interaction potential Φvdw of BSA particles in HFA 227.

[0154]FIG. 46 is a table of the settling behavior of BSA particles
prepared by TFF, milling, and spray drying and calculations for porous
shell particles prepared by spray drying, with the aValue determined
from the equivalent volume of a sphere measured from laser light
scattering; bThe density difference was determined by ρf-ρL
with ρp=1.5 g/cm3; cDetermined from dimensions given by
Dellamary et al.; dCalculated for primary particle with 100 nm thick
shell. FIG. 47 is an optical image of protein pMDI formulations (Lys in
HFA 227 with a drug loading of 20 mg/mL, Lys in HFA 134a with a drug
loading of 40 mg/mL, 50 mg/mL, 90 mg/mL, and BSA (BSA) in HFA 227 with a
drug loading of 50 mg/mL, left to right) 4 hours after shaking FIG. 48 is
a SEM micrograph of aerosolized Lys particles (Lys in HFA 134a pMDI
loaded at 50 mg/mL). Aerosolized particles have geometric diameters
between 8-10 μm (A) and exhibit porous morphology (B) and (C).

[0155]This invention is a new composition of matter by process for
producing highly concentrated (about 10-90 mg/mL), suspensions of drugs
in pressurized metered dose inhalers (pMDIs). This approach may be used
for many types of low molecular weight drugs, and for high molecular
weight drugs including peptides and proteins. Dry powders of submicron
protein particles produced by thin film freezing, a powder formation
process described in manuscripts by Overhoff et. al and Engstrom et. al.
(incorporated herein), readily disperse when added to a hydrofluoroalkane
propellant to form a stable suspension.

[0156]Upon actuation, the submicron protein particles contained within the
propellant droplets aggregate to form a porous protein structure (e.g.,
8-10 μm) ideal for pulmonary deposition. Pulmonary delivery of
proteins is of great interest because the lungs are far more permeable to
macromolecules compared to other routes into the body, such as the
gastrointestinal (GI) tract, and less invasive than parenteral routes.
Furthermore, lung concentrations of metabolizing enzymes are lower than
that found in the GI tract and liver.

[0157]At concentrations in an HFA of 10 mg/mL achieved emitted and
respirable doses of 700 μg and 300 μg per actuation, respectively,
of bovine serum albumin (BSA). The new work extends this concept to
concentrations of up to 90 mg/mL in HFA 134a. This leads to emitted doses
as high as 4 mg/actuation as described below in TABLE 5. One of the
primary criticisms levied against pMDI formulations is the upper limit
dose that they can deliver, 500-600 μg/dose. Common pMDI doses are
100-300 μg/dose5. Thus, a major goal for pMDIs has been to raise
the dosage in order to allow for pMDI delivery of less potent actives.

[0158]To prepare the protein pMDI formulations, the protein powders are
placed in 50 mL PYREX® beakers and pre-cooled in a -80° C.
freezer. The propellant, preferably a hydrofluoroalkane propellant, is
pre-cooled to -80° C. and then poured into the beaker containing
the protein powders to form suspensions ranging in concentrations from
0.7-7.4% w/w. The resultant protein suspensions are placed in a dry
ice/acetone bath and sonicated for two minute using a Branson Sonifier
450 (Branson Ultrasonics Corporation, Danbury, Conn.) with a 102
converter and tip operated in pulse mode at 35 W. 11 mL of the cooled
protein formulations are dispensed into 17 mL glass aerosol vials (SGD,
Paris, France) and fitted with metering valves containing 100 μL
metering chambers (DF10 RC 150, Valois of America, Inc., Congers, N.Y.)
using a compressor pump (Pamasol Model P2005, Pfaffikon, Switzerland).
The vials are allowed to warm up to room temperature. Small amounts of
lubricants (2-8% w/w), such as polysorbate 20 and polysorbate 80, may be
added to the formulation prior to sonication to minimize clogging of the
valve during actuation due to the highly concentrated suspensions.

[0159]The highly concentrated protein pMDI's demonstrate desirable
aerodynamic properties ideal for pulmonary drug delivery. Impaction
studies were conducted with a non-viable eight-stage cascade impactor
(Thermo-Andersen, Smyrna, Ga.) with an attached Aerochamber Plus®
Valved Holding Chamber (Trudell Medical International, London, Ontario,
Canada) at a flow rate of 28.3 L/min to quantify total emitted dose
(TED), fine particle fraction (FPF), mass median aerodynamic diameter
(MMAD), and geometric standard deviation (GSD). FPF was defined as the
percentage of particles with an aerodynamic diameter less than 4.7 μm.
Three actuations are expelled as waste prior to measurements. One
actuation is made into the ACI for analysis. The valve stem, actuator,
and impactor components are placed into separate containers with a known
volume of deionized (DI) water. Each component soaks for at least 30
minutes to ensure complete protein dissolution. The protein
concentrations are quantitated with a Micro BCA Protein Assay
manufactured by Pierce (Rockford, Ill). The absorbance of the solutions
was measured at 562 nm using the μQuant Model MQX200 spectrophotometer
(Biotek Instruments Inc., Winooski, Vt.). Untreated protein was used to
prepare the protein standards at concentrations between 2 and 40
μg/mL. ACI results yielded average TED's between 2.5-4.0 mg
protein/actuation, average FPF's between 56-64%, and average MMAD's
between 2.5-2.8 (GSD's between 3.0-3.9).

[0160]The ability of a pMDI to deliver a consistent dose is mandatory for
the delivery of sufficient and safe drug doses to patients. Thus, stable
pMDI suspensions are desired to ensure dose uniformity. No visible
creaming or settling was observed for the suspensions over 48 hours. FIG.
47 is an optical image of protein pMDI formulations (Lys in HFA 227 with
a drug loading of 20 mg/mL, Lys in HFA 134a with a drug loading of 40
mg/mL, 50 mg/mL, 90 mg/mL, and BSA (BSA) in HFA 227 with a drug loading
of 50 mg/mL, left to right) 4 hours after shaking.

[0161]Dose uniformity of the highly concentrated protein pMDI's is
demonstrated by actuating the pMDI through the firing adaptor of a dosage
unit sample tube (26.6×37.7×103.2 mm; 50 mL volume; Jade
Corporation, Huntingdon, Pa.). A known volume of DI water is added to
dissolve the protein and the sampling tube is shaken and allowed to sit
for at least 30 minutes to ensure complete protein dissolution. The
protein concentration is determined using the Micro BCA protein assay in
conjunction with the μQuant spectrophotometer. The pMDI canister is
weighed before and after each actuation to assure that the proper dose
was released.

[0162]TABLE 6 is a table of the dose uniformity results for different
protein pMDI formulations at different protein concentrations. %
Theoretical is the percentage of the theoretically loaded dose that is
emitted during actuation.

[0163]Further characterization of protein particles after aerosolization
from the pMDI device is performed. The aerosolized particles are measured
by laser light scattering. Each formulation is actuated once through the
ACI spacer and throat. The aerosol exits the outlet of the throat about 5
cm above the laser of the Malvern Mastersizer S (Malvern Instruments,
Ltd., Worcestershire, UK). For each formulation 100 measurements of the
aerosolized spray are made about every 5 ms. The recorded measurements
are averaged to give a final profile of the aerosolized particles on a
volume basis. Scanning electron microscopy (SEM) images of aerosolized
particles are also used to determine the size of aerosolized particles.
Particles are collected from stage 3 of the ACI. Double carbon adhesive
tape is applied to stage 3. The impaction test is conducted according to
the parameters mentioned earlier and the carbon tape is applied to an
aluminum SEM stage. The sample is sputter coated with gold-palladium for
30 seconds using a K575 sputter coater (Emitech Products, Inc., Houston,
Tex.). Micrographs are taken using a Hitachi S-4500 field emission
scanning electron microscope (Hitachi Ltd., Tokyo, Japan) at an
accelerating voltage of 5-10 kV. Particle images are sized on the SEM
micrographs using imaging software (Scion, Frederick, Md.). At least 50
particles were measured for each formulation. Particle sizes from the SEM
micrographs correlate well with sizes reported by laser light scattering.

[0164]FIG. 48 is a SEM micrographs of aerosolized Lys particles (Lys in
HFA 134a pMDI loaded at 50 mg/mL). Aerosolized particles have geometric
diameters between 8-10 μm (FIG. 48A) and exhibit porous morphology
(FIG. 48B) and (FIG. 48C). Other formulations show similar morphologies.
Aerosolized particle densities are also determined from the SEM
micrographs. The calibrated aerodynamic diameter of particles deposited
on stage 3 of the ACI is 3.3-4.7 μm. Thus an average MMAD of 4.0 μm
was assumed for particles deposited on stage 3 of the ACI. Using the
relationship, da=dg(ρg/ρa)0.5 (where
da is the aerodynamic diameter, dg is the geometric diameter,
ρg is the density of the particle, and ρa is 1
g/cm3) and using the estimated MMAD and geometric diameter (from the
SEM micrographs), the density of the aerosolized particle is calculated.
The low calculated densities (0.14-0.23 g/cm3) indicate that the
aerosolized particles are highly porous, which is expected because of the
porous morphology observed in the SEM micrographs. The low densities
explain why the particles are able to reach deep lung levels despite a
geometric diameter of 8-10 μm.

[0165]TABLE 7 is a table that illustrates the measured particle diameters
for aerosolized protein particles. Dv,50 (diameter at which the
cumulative sample volume was under 50%) values were reported by Malvern.

[0166]Preparation of TFF ITZ particles. ITZ (about 500 mg, Hawkins, Inc.,
Minneapolis, Minn.) was dissolved in about 40 mL of 1,4-dioxane (Fisher
Chemicals, Fairlawn, N.J.). To the drug solution, 100 mL of t-butanol
(Fisher Chemicals, Fairlawn, N.J.) was added. The ITZ in 1,4
dioxane-t-butanol drug solution was passed at a flow rate of 4 mL/min
through a 17 gauge (1.1 mm ID, 1.5 mm OD) stainless steel syringe needle.
The droplets fell from a height of 10 cm above a rotating stainless steel
drum (12 rpm) 17 cm long and 12 cm in diameter. The hollow stainless
steel drum was filled with dry ice to maintain a drum surface temperature
of about 223 K. On impact, the droplets deformed into thin films and
froze. The frozen thin films were removed from the drum by a stainless
steel blade and transferred to a 400 mL PYREX® beaker filled with
liquid nitrogen. The excess liquid nitrogen was evaporated in a
-80° C. freezer. A Virtis Advantage Lyophilizer (The Virtis
Company, Inc., Gardiner, N.Y.) was used to dry the frozen slurries.
Primary drying was carried out at -30° C. for 36 hours at 300
mTorr and secondary drying at 25° C. for 24 hours at 100 mTorr. A
12 hour linear ramp of the shelf temperature from -30° C. to
+25° C. was used at 100 mTorr.

[0167]Crystallization of TFF ITZ particles: FIGS. 49A-49C are optical
images of the TFF ITZ particles (about 110 mg) were loaded as dry powder
into glass vials (SGD, Paris, France) and fitted with metering valves
(DF10 RC 150, Valois of America, Inc., Congers, N.Y.) using a Pamasol
Model P2005 compressor pump (Pamasol Willi Mader A G, Pfaffikon,
Switzerland) (FIG. 49A). FIG. 49A is a photograph of 110 mg TFF ITZ
powder loaded into a glass vial. FIG. 49B is a photograph of a 10 mg/mL
TFF ITZ suspension produced after addition of 11 mL of HFA 227 to FIG.
49A, and FIG. 49C is a photograph of 110 mg TFF ITZ powder after exposure
to HFA 227. 1,1,1,2,3,3,3-heptafluoropropane (HFA 227, Solvay, Greenwich,
Conn.) was loaded into the vials containing drug using Pamasol filling
equipment (Model P2008) to yield a 10 mg/mL milky suspension (FIG. 49B).
The pressurized suspensions may be referred to as pressurized metered
dose inhalers (pMDIs). To collect TFF ITZ powder after HFA exposure, the
pMDI was cooled in a -80° C. freezer, well below the HFA 227
boiling point of -16° C. Once the HFA was sufficiently cooled, the
metering valve was removed and the HFA was allowed to warm in a dry box
(relative humidity<20%) until it completely evaporated (FIG. 49C).
Exposure of TFF ITZ powder to 2H,3H perfluoropentane (HPFP), a
non-volatile surrogate for HFA 227, was also studied.

[0168]Product Description and Characterization: Sub-micron amorphous
particles of a poorly water soluble drug, itraconazole (ITZ), were
produced by thin film freezing (TFF), a particle formation process
described in manuscripts by Engstrom et al. and Overhoff et al.

[0169]FIG. 50 is a graph of the X-ray diffraction (XRD) pattern of ITZ
before and after exposure to HFA 227. Crystallization of TFF ITZ
particles after HFA exposure was determined using x-ray diffraction (XRD)
and differential scanning calorimetry (DSC). XRD patterns and DSC scans
of TFF ITZ powder before contact with HFA were characteristic of
amorphous materials (FIGS. 50-51). However, characteristic peaks of
crystalline ITZ were detected in the XRD profile after TFF ITZ particles
were exposed to HFA 227 (FIG. 50).

[0170]FIG. 51 is a graph of the Modulated differential scanning
calorimetry (mDSC) of TFF ITZ powders before and after exposure to HFA
227 and HPFP and pure ITZ. DSC scans showed complete crystallization of
the TFF ITZ particles after exposure to HFA 227, based on the absence of
an endothermic recrystallization peak (FIG. 51). Similar results were
obtained after exposure of TFF ITZ particles to HPFP (FIGS. 50-51). These
results are significant because crystallization of TFF ITZ may be induced
with a solvent that can be handled under atmospheric conditions.

[0171]To confirm the complete crystallization of TFF ITZ particles after
exposure to HFA, dissolution studies were conducted on the HFA-exposed
TFF ITZ powder in pH 7.4 phosphate buffer (0.02% w/v SDS). The
equilibrium solubility of crystalline ITZ in the dissolution media was
experimentally determined to be 1.4 μg/mL. HFA-exposed TFF ITZ powder
(1 mg) was added to 50 mL of dissolution media to yield an initial drug
loading of 20 μg/mL. Sample aliquots (1.5 mL) were taken from the
dissolution vessels at various time points. The aliquots were filtered
immediately using a 0.2 μm syringe filter. Dissolved drug levels did
not significantly exceed equilibrium solubility of crystalline ITZ,
suggesting that the HFA-exposed TFF ITZ particles were crystalline (FIG.
52). FIG. 52 is a graph of the dissolution profile of TFF ITZ particles
after exposure to HFA 227 conducted in pH 7.4 phosphate buffer (0.02% w/v
SDS). TFF ITZ powder that had been previously exposed to HFA 227 (1 mg)
was added to the dissolution media (50 mL) to achieve an initial loading
of 20 μg ITZ/mL. All samples were filtered with 0.2 μm pore size
filters. The dashed line represents the solubility of "as received" ITZ
in the dissolution media.

[0172]FIG. 53 in a scanning electron microscopy (SEM) images of TFF ITZ
(FIG. 53A) before and (FIG. 53B) after exposure to HFA 227 and (FIG. 53C)
SEM image of TFF ITZ after pMDI was actuated into water, without any
exposure to air. Additionally, a change in morphology of the TFF ITZ
particles before and after exposure to HFA 227 was detected by scanning
electron microscopy (FIG. 53A-53B). TFF ITZ particles prior to HFA
contact were spherical in shape. However, thin, plate-like structures
were observed after exposure to HFA. To further verify that the
crystallization of TFF ITZ was induced by HFA, a pMDI containing TFF ITZ
was actuated into water, with the metering valve submerged below the
liquid level, to produce a slightly turbid dispersion. The TFF ITZ
particles emitted from the pMDI were collected by freezing and
lyophilizing this dispersion. SEM images of the actuated TFF ITZ
particles revealed thin, plate-like structures strongly resembling the
particles produced after HFA evaporation (FIG. 53C). Therefore, complete
crystallization of amorphous TFF ITZ particles occurred upon exposure to
HFA 227.

[0173]FIG. 54 is a graph of the dynamic light scattering (DLS)
measurements of HFA-exposed TFF ITZ in water. The sizes of 66% of the
particles (by volume) were 737 nm or less. Furthermore, dynamic light
scattering (DLS) measurements show that TFF ITZ particle dimensions
remained below 1 μm after crystallization, with 66% of the particles
by volume with a hydrodynamic radius of 737 nm or less (FIG. 54).

[0174]Production of TFF ITZ/BSA compositions. Two compositions using a
10/1 and 5/1 ITZ/BSA ratios were formulated to demonstrate that a water
soluble component could be added to the poorly water soluble TFF ITZ to
aid in wetting during dissolution. A 5 mg/mL loading was tested. The
resultant pMDI formulations were milky, white and uniform, and similar to
the other TFF pMDIs.

[0177]FIG. 56 is a plot showing the dissolution profiles of aerosolized
TFF ITZ and aerosolized milled ITZ particles (300 nm) studied in
phosphate buffer (pH=7.4) containing 0.2% w/v SDS at 37° C. The
graph shows a much more rapid dissociation and dissolution of the
aerosolized aggregate into constituent particles in comparison to the
milled ITZ particles.

[0178]The flocculated particles used for pMDI delivery may also be
applicable for dry powder inhalation. In a dry powder inhaler, shear
forces generated during inspiration break up the flocs to an appropriate
aerodynamic size for deep lung delivery. The particles may be produced by
either milling, controlled precipitation (CP), or TFF. Poorly water
soluble drugs, itraconazole (ITZ) and cyclosporine A (CsA), and water
soluble proteins, bovine serum albumin (BSA) and lysozyme (lys), were the
model drugs used to demonstrate DPI delivery of nanoparticles produced by
CP and TFF. Drug powders were aerosolized and characterized using either
an Aerosizer/Aerodisperser (TSI, Shoreview, Minn.) or an APS 3321/3343
(TSI, Shoreview, Minn.) disperser.

[0179]FIG. 57 is a graph of the aerodynamic diameters of milled, TFF, and
CP drug compositions measured by the APS 3321/3343 and the
Aerosizer/Aerodisperser systems. VMAD is the volume mean aerodynamic
diameter. The drug compounds studied include the poorly water soluble
drugs itraconazole (ITZ) and cyclosporine A (CsA), as well as bovine
serum albumin (BSA) and lysozyme (lys). T80 and T20 are the surfactants
tween 80 and tween 20 (Sigma Chemical, St. Louis, Mo.).

[0180]The aerosolized CP and TFF powders possessed aerodynamic diameters
predominantly between 2.0-3.5 μm, on a volume basis, ideal for
pulmonary delivery, as seen in FIG. 57. These diameters are in a range
that is known to be desirable for efficient deep lung delivery.
Furthermore, the sizes are in good agreement for the two dispersers. Only
two compositions containing low melting point stabilizers, such as Tween
surfactants, possessed aerodynamic diameters larger than 8 μm.
Compositions containing both a poorly water soluble drug (Itz) and a
protein (BSA) were also shown to yield optimal aerosol particles for
pulmonary delivery.

[0181]FIG. 58 is a graph of the aerodynamic particle size distribution for
the TFF lys composition. VMAD is the volume averaged mean aerodynamic
diameter and GSD is the geometric standard deviation.

[0182]An example of the aerodynamic particle size distribution for the
aerosolized particles is shown in FIG. 58 for the aerosolized TFF lys
formulation. SEM micrographs of TFF lysozyme powder before aerosolization
and after aerosolization are shown in FIG. 59A-C. Lysozyme particles
produced by TFF have a morphology of small nanorods, with lengths
˜500 nm and diameters between ˜50-100 nm, as seen in FIG.
59A-C. Aerosolization of the powder disperses the nanorod floc to yield
aerosol particles roughly 3 micron in diameter. High magnification images
of the aerosolized particles show that the rod-shaped primary particles
are maintained throughout the aerosolization process. The SEM micrographs
of the aerosolized TFF lys particles were obtained by placing ˜25mg
of powder in gallon sized Ziploc bag. Double sided carbon tape was placed
onto the inside of the bag. The opening of the bag was then rubber banded
around the nozzle of a can of compressed air. A short burst of air was
actuated into the bag to disperse the powder. The carbon tape was removed
from the inside of the bag and placed onto an SEM stage for microscopy.

[0183]The present invention provides a novel composition and method of
making compositions for the development of a dry powder inhaler (DPI)
system comprised of highly dispersible and deformable nano-structured
aggregates that may be templated by air to form aerosol particles
appropriate for pulmonary delivery. The nano-structured aggregates
consist of a porous web of drug nanoparticles, in which the primary
particles making up the web are touching one another but are not tightly
packed. The sizes of the individual webs may range from several microns
to several hundred microns, as observed by scanning electron microscopy.
The primary drug particles may be spherical (aspect ratio near or equal
to 1) or elongated (aspect ratio greater than 1) in shape. The
nanoparticle web is considered deformable because, upon entrainment in
air, portions of the nanoparticle web may be sheared off by design into
smaller aggregates. These smaller aggregates possess aerodynamic
diameters appropriate for deep lung delivery (2-5 μm), as determined
by time-of-flight measurements. Thus, the final aerosolized particle is
"templated by air," as the air stream provided by a patient's inspiration
through a DPI device is capable of providing the force necessary to shear
off a portion of the nanoparticle web to form an inhalable particle.
Highly dispersible and deformable nano-structured aggregates of
itraconazole (ITZ), bovine serum albumin (BSA), and ITZ/BSA nanoparticles
have been produced. This approach is applicable to other proteins, gene
delivery, peptides and low molecular weight drugs of a range of water
solubilities.

[0184]Until recently, the delivery of protein therapeutics has been
largely limited to parenteral delivery due to the chemical and physical
instabilities of proteins and challenges in permeating biological
membranes (79) Among the non-invasive routes, pulmonary delivery offers
advantages of large alveolar surface area (˜100 m2), rapid
absorption across the thin alveolar epithelium (0.1-0.5 μm), avoidance
of first pass metabolism, and sufficient bioavailabilities (79-87). DPIs
are the newest form of pulmonary mode of administration of active
pharmaceutical ingredients and have become a popular delivery method for
drugs, especially proteins, because delivery and storage as a dry powder
is desirable in terms of stability. Because optimal aerodynamic
properties are crucial to deep lung deposition, particle aggregation and
inefficient powder dispersion, common problems observed in DPIs, are
detrimental to DPI performance. Attempts to optimize particle
interactions within a DPI formulation have included the addition of
carrier particles to improve powder flow properties, and the modification
of particle shape, size, surface roughness, or surface energy by the
addition of low surface energy particles (88).

[0185]The nano-structured aggregates readily deform and disperse into
optimally sized particles for pulmonary delivery upon entrainment with
air without the need for carrier particles and minimal amounts, and in
some cases, no surfactant, which facilitates the production of high
potency drug powders. Traditional DPI powders consist of pre-formed 1-10
μm particles. To achieve high fine particle fractions (FPF), the DPI
device must efficiently deaggregate the pre-formed powder particles down
to primary particles. Particle properties such as shape and surface
roughness strongly influence dispersion characteristics, and consequently
FPF and aerodynamic properties, of drug powders from a DPI (88).
Furthermore, traditional DPI particles typically experience strong
attractive Van der Waals (VDW) forces when packed into a DPI device due
to short separation distances between particles (estimated to be on the
order of 5 nm (89)), which impedes efficient particle deaggregation.
Unlike conventional DPIs, this invention does not require the DPI device
to deliver pre-formed powder particles comprising drug, but more
advantageously aggregates of the primary drug particles. By loading a
porous web of nanoparticles into a DPI device, common DPI problems
observed with pre-formed particles, such as mechanical interlocking and
high surface energies between particles, are minimized due to the porous
structure of the nanoparticle aggregates, which experience a reduced
attractive VDW force compared to dense micron-sized particles (89-91).
Thus, the nano-structured aggregates are easily sheared and deformed into
respirable aggregates with aerodynamic diameters (da) between 2-5
μm due to the extremely weak Van der Waals forces holding the porous
aggregate together.

[0186]This invention provides an efficient way to deliver nanoparticles to
the lungs using a DPI. Optimal aerodynamic behavior has been achieved by
delivering highly dispersible and deformable, porous aggregates of
nanoparticles as a dry powder for enhanced pulmonary delivery. Pulmonary
delivery to replace other methods such as parenteral and oral delivery.

[0187]There are possible challenges concerning the stability of the drug
powder in a compacted state (when loaded into a DPI device) over long
periods of time. Long term storage may cause aggregation which may affect
the ability of the powder to efficiently disperse into aerosol particles
optimal for pulmonary delivery. Minimal aggregation between particles is
anticipated for the porous nanoparticle aggregates, due to weak
attractive VDW forces, which results from their porous structure. Strong
attractive VDW forces between particles increase the possibility for
irreversible aggregation, a problem for conventional DPIs (88). In the
case that the drug particles aggregate and do not disperse efficiently
after storage, components such as leucine may be added to the
formulation, which has been shown to enhance dispersability of dry
powders for inhalation(88,92).

[0188]Dry powder inhaler (DPI) formulations typically consist of
micronized drug blended with carrier particles packed into a bed (e.g.,
capsule, blister pack, etc). In order to achieve effective drug
deposition in the lungs, the patient must generate sufficient shear and
turbulence to fluidize the packed powder and carry the drug particles to
the lungs upon inspiration. Thus, the ability of the packed drug
particles to disperse into primary particles is essential for the
aerosolized powder to possess optimal aerodynamic properties for deep
lung deposition. To enhance particle dispersion in DPIs, Langer and
Edwards have produced large, porous particles (AIR® particles), with
diameters between 5-20 μm and low tap densities (<0.4 g/cm3),
which experience reduced Van der Waals attractive interactions compared
to non-porous particles due to their porous morhpology. Despite the large
size of AIR® particles, their low porosity allows them to possess
aerodynamic properties similar to that of smaller, non porous particles.

[0189]In this invention, nano-structured aggregates have been shown to
readily disperse into optimally sized particles for pulmonary delivery
upon entrainment with air without the need for carrier particles and
minimal amounts, and in some cases, no surfactant, which facilitates the
production of high potency drug powders. Unlike traditional micronized
DPI powders and AIR® particles, this invention does not require the
DPI device to deliver primary drug particles, but aggregates of the
primary drug particles. The porous web of nanoparticles is easily sheared
into aggregates with aerodynamic diameters (da) between 2-5 μm
due to the extremely weak Van der Waals forces holding the porous
aggregate together.

[0190]The present invention provides a method of making brittle-matrix
particles through blister pack freezing using ultra-rapid freezing (URF)
technology adapted for manufacture in a pharmaceutical blister pack. For
example, the present invention can use ultra-rapid freezing using the
blister packs contents and an ADVAIR DISKUS®. The device was opened,
the blister strip was removed and peeled open. The contents (drug and
lactose) of each blister pack were removed. The aluminum strip was
cleaned with deionized water, rinsed with ethanol, and allowed to dry and
room temperature.

[0191]The brittle-matrix particles were formed. Tacrolimus and lactose
(TACLAC) and tacrolimus (TAC) solutions were prepared separately in 1 mL
of ACN:water (3.2) each. Both solutions contained 0.75% w/v solids. A
PYREX® Petri dish was filled with liquid nitrogen, and blister packs
were added. One at a time, blister packs were removed from the liquid
nitrogen bath, and 25 μL of drug solution was added to the concave
indentation. The product was frozen immediately upon contact and placed
in a -80° C. freezer. All frozen blisters were lyophilized
according to the recipe described herein. After lyophilization, all
blister packs were stored under vacuum in a sealed desiccator.

[0192]Aerosol testing was conducted using a Next Generation Pharmaceutical
Impactor (NGI) with coated collection surfaces. Carefully, a single
blister containing either TACLAC or TAC was added to the inhalation
position of the ADVIAR DISKUS®, and the device was resealed and
mounted on the induction port of the NGI by a silicone molded fitting.
Flow rate necessary to achieve a 4 kPa pressure drop across the Diskus
was determined to be 66 L/min; therefore, all studies were conducted at
this flow rate. Both TACLAC and TAC blister formulations were actuated
three times before collection of the impacted formulation from the
stages. Rinsing and high performance liquid chromatography (HPLC) method
for quantification of drug was performed. Fine particle fraction is
defined as the percentage of drug mass emitted that is below 5 μm in
diameter.

[0193]FIG. 60 is an aerodynamic distribution of brittle-matrix particles
emitted from an ADVAIR DISKUS®. FIG. 60 shows the aerodynamic
distribution of both formulations analyzed and the FPF measured for
TACLAC and TAC formulations were 35.1% and 19.8%, respectively. Clearly,
the shear imparted by the Diskus device is not sufficient to obtain the
quantity of highly respirable particles made by the HANDIHALER®.
Although FPFs measured in this initial study were low in comparison,
brittle-matrix particles tested here still outperformed the formulations
marketed with the Diskus. The FLUTIDE DISKUS® was evaluated for
efficiency in a study by Steckel in 1997, where only 25.4% of the emitted
dose was in the aerodynamic range below 6.4 μm (92). TACLAC, when
prepared by blister freezing, resulted in an aerosol with 41.0% of the
emitted dose below 6.4 μm.

[0194]The total emitted dose (TED) for TACLAC and TAC formulations were
78.6% and 97.3%, respectively. It was apparent during formulation
production that temperature of the blisters packs determined the shape
and morphology of the frozen formulation because of the effect on the
rate of freezing. This may have contributed to some of the difference in
TED between formulations, as TACLAC blisters were thought to be warmer
upon addition of the drug solution. It was also observed that even upon
storage in a vacuum desiccator, the hydroscopic effects of lactose caused
"collapse" of TACLAC particles. Cohesion caused by moisture sorption
could also have caused increased retention of TACLAC in the blister.

[0195]As used herein, the term "surfactant" means a substance that reduces
the surface tension of a liquid, thereby causing it to spread more
readily on a solid surface. Examples of surfactants for use with the
present invention, include, all surfactants suitable for administration
to the lungs, including sodium salts of cholate, deoxycholate,
glycocholte and other bile salts; Span 85, Lauryl-beta-D-maltoside,
palmitic acid, glycerol trioleate, linoleic acid, DPPC oleyl alcohol,
oleic acid, sodium oleate, and ethyl oleate.

[0197]Delivery of the present invention to the lung can be achieved
through any suitable delivery means, including a nebulizer, a dry powder
inhaler, a metered dose inhaler or a pressurized metered dose inhaler.
The suitable delivery means will depend upon the active agent to be
delivered to the lung, the desired effective amount for that active
agent, and characteristics specific to a given patient.

[0200]The preparation of particles and respirable aggregates using a URF
method includes a solution of ITZ (0.0798 g) with pluronic F-127 (0.0239
g) is prepared by loading the dry solids into a vial. A prepared 95/5 wt
% blend of t-butanol and toluene (10.03 g) is loaded into the vial. The
resulting slurry is heated until a solution was formed. (68 to 70.
degree. C.). The resulting solution is applied to the freezing surface of
the URF unit, which had been cooled to -78° C. over a three-minute
time period. The frozen solvent, drug, and excipient matrix is collected
in a tray, which had been cooled with dry ice, and transferred into a
60-mL jar, which had been cooled with dry ice. The jar containing the URF
processed frozen solid is then placed on a freeze drying unit and
lyophilized for approximately 17 hr at 100 mtorr. After lyophilization,
0.0700 g of the URF processed solid is recovered as a dry flowable
powder. The mean volume average particle sizes (with and without
sonication) of the reconstituted drug particles are measured using a
Coulter LS 230. The particles are amorphous.

[0201]Pulmonary inhalation of low-density porous particles enables deep
lung delivery with a more efficient dose and less dependence on device
design and patient inspiration. The large geometric diameter of porous
particles enhances sustained in vivo drug release by avoidance of
physiological clearance mechanisms. The present invention provides
respirable low-density microparticles (25-50 μm) produced in situ from
brittle drug matrices to achieve highly efficient deep lung delivery via
a dry powder inhaler. The brittle matrices comprising a solid dispersion
of drug and excipient are sheared apart by a standard inhalation device
to produce ultra low-density particles with appropriate aerodynamic
diameters (1-5 μm). Skeletal particle density for each formulation
determined from the measurement of the geometric and aerodynamic
diameters were as low as 0.01 g/mL. In contrast, reported skeletal
densities of large porous particles produced by other techniques are
>0.05 g/ml and often >0.1 g/ml After incorporation of biocompatible
materials such as pharmaceutical sugars into the formulations,
aerosolization of the resulting brittle matrices produced fine particle
fractions (FPF) as high as 70.3% and total emitted doses (TED)
consistently higher than 95%. Accuracy of aerodynamic testing with
cascade impaction was improved markedly by coating the collection
surfaces. The aerosolization of the particles was found to be susceptible
to humidity induced capillary forces and electrostatic charging, although
formulations containing mannitol or no sugar excipient proved to be more
robust. Under completely dry conditions, the formulation made with
anhydrous lactose exhibited improved brittle fracture and aerosolization,
showing a 10% increase in FPF and 0.8 μm decrease in mass median
aerodynamic diameter (MMAD) relative to the same formulation stored at
50% RH. Low-density microparticles, produced from aerosolization of
brittle matrices produced by thin film freezing (TFF) exhibit exceptional
respirable properties and may prove to be a useful platform for highly
efficient delivery of thermally labile, highly potent, and poorly soluble
drugs.

[0202]TFF technology was employed for the production of dry powders.
Briefly, a cosolvent mixture of acetonitrile (ACN) and water was used to
dissolve tacrolimus and sugar excipient. Tacrolimus and lactose (TACLAC),
tacrolimus and mannitol (TACMAN), tacrolimus and raffinose (TACRAF), and
tacrolimus without a sugar excipient (TAC) were dissolved in the
cosolvent solution. The ratio of tacrolimus to excipient was 1 to 1 and
each solution prepared for TFF had a total solids concentration of 0.75%
w/v. The solutions were rapidly frozen on a cryogenically cooled
(<-50° C.) stainless steel surface and then maintained in the
frozen state in liquid nitrogen. A detailed description of the TFF
process is given by Overhoff et al and Engstrom et al. Solvents were
sublimated by lyophilization using a VirTis Advantage Tray Lyophilizer
(VirTis Company Inc., Gardiner, N.Y.), leaving a drug and sugar solid
dispersion in dry low-density particles. Lyophilization was performed
over 40 hours at pressures less than 200 mTorr while the shelf
temperature was gradually ramped form -60° C. to 25° C.
Product was removed form the lyophilizer after dry N2 was bled into
the chamber to equilibrate to atmospheric pressure. Product was quickly
covered in order to prevent ambient humidity from affecting the
formulation. Powders were stored in a transparent vacuum desiccator at
room temperature.

[0203]Bulk and tapped density of TFF produced powders were measured
according to a method adapted from USP method I using a Varian Tapped
Density Tester (Varian, Palo Alto, Calif.). An adaptation was made due to
the limited supply of powder for testing where a 100 mL graduated
cylinder was replaced by a 5 mL graduated cylinder. Hausner ratio and
Can's (Compressibility) index were calculated for each formulation based
on USP guidelines. Additionally, skeletal densities of dispersed powders
were calculated based on measured aerodynamic and geometric diameter for
comparison to measured density values. Calculations were performed, where
the dynamic shape factor (X) was assumed to be 1.5 for all dispersed
powders. Mass median aerodynamic diameter (MMAD) was determined based on
all particles emitted from the device for these calculations.

[0204]Geometric diameter of TFF produced aerosolized and non-aerosolized
powder was determined by low angle light scattering with an inhalation
cell and an induction port. A HANDIHALER® (Boeringher Ingelheim GmbH,
Ingelheim am Rhein, Germany) containing a size 3 hypromellose (HPMC)
capsule was secured to the mouth of the induction port by a molded
silicone adapter. Aerosolization of powder was achieved at a flow rate 51
L/min, providing a 4 kPa pressure drop across the device. Data
acquisition took place over 4 seconds and only when laser transmission
dropped below 95%. Non-aerosolized powder diameter was measured by adding
powders to the opening of the inhalation cell without the induction port
and without air flow.

[0205]A Next Generation Pharmaceutical Impactor (NGI) (MSP Corp.,
Shoreview, Minn.) was used to determine aerodynamic properties of
low-density microparticles. A HANDIHALER® containing size 3 capsules
and approximately 3 mg of formulation was attached to the induction port
by a molded silicone adapter. All tests, with the exception of those
investigating the influence of gelatin capsules on aerodynamic diameter,
were conducted with size 3 HPMC capsules. Aerosols were produced over 4
seconds at a flow rate of 51 L/min. Stage cut size diameters were
calculated to be 8.8, 4.9, 3.1, 1.8, 1.0, 0.6, 0.4, and 0.2 μm for
stages 1 through 7 and micro-orifice collector (MOC), respectively (24).
In most impaction tests run, collection surfaces were coated with 1%
Tween 80 in ethanol, which is one of many coating materials recommended
by the European Pharmaceutical Aerosol Group (EPAG). Tween solution was
applied to each collection surface (approx 1 mL) and allowed to dry for 1
hour. After aerosolization, collection of deposited powders was
accomplished by rinsing with 2, 5, 10, and 2.5 mL mobile phase for the
device, induction port, pre-separator (if used), and stages 1-MOC,
respectively. The pre-separator is designed to collect coarse particles
(>15 μm) before the enter the body of the NGI and was included only
when coarse lactose is used. High performance liquid chromatography
(HPLC) and a method for tacrolimus detection were used to quantify the
collected drug from each rinsing.

[0206]Total emitted dose (TED) of each test was calculated as the
percentage of dose emitted over total dose assayed. Fine particle
fraction (FPF) and MMAD were calculated using Sigmaplot 2000 (Systat
Software Inc, San Jose, Calif.) to fit a 3 parameter logistic curve to
plotted data. MMAD and geometric standard deviation (GSD) were calculated
based on drug deposition on stage 1 through MOC, while FPF was calculated
based on TED and represent the percentage of particles with an
aerodynamic diameter less than 5 μm.

[0207]Water sorption profiles were determined for brittle matrix powders
manufactured by TFF using Dynamic Vapor Sorption (DVS-1). For each
formulation, glass sample cells were filled to capacity (0.5 mL)
resulting in weights ranging from 5 to 30 mg, depending of particle
density. Samples were dried with nitrogen gas until a baseline was
established with less than 0.002% change in dm/dt. Each formulation was
run for a complete sorption/desorption cycle between 0 and 90% relative
humidity (RH). Humidity was increased/decreased by 5% after equilibrium
was reached, as determined by a dm/dt less than 0.002%. Sorption
isotherms were calculated and plotted according to percent change in mass
minus the initial dry formulation weight. DVS was also used to create a
controlled humidity environment for powder dispersion to be tested using
laser scatter. Humidities of 90, 50, 20, and 0% were exposed to powder
formulations for 30 minutes in succession. Equilibrium was assumed after
30 minutes, and an aliquot of powder was removed for testing. All testing
began with 90% humidity so that skeletal density changes due to
hygroscopicity would be applied to subsequent samples taken at 50, 20,
and 0% RH.

[0208]TFF technology produces low-density pharmaceutical matrices, often
containing amorphous drug, stabilized with high Tg excipients. In
previous reports, TFF has been used as a particle engineering technology
to enhance the aqueous solubility of poorly water soluble drugs for oral
and pulmonary applications. Through stabilization of amorphous drug
morphologies with glassy excipients, inclusion of hydrophilic materials,
and increased surface area, TFF manufactured powders have been shown to
offer improvements in wetting, dissolution rates, and solubility, leading
ultimately to increased bioavailability. Given the desirable attributes
of these powders and the efficiency of low-density powders for deep lung
delivery, we hypothesized that these particles would result in superior
aerosol performance relative to previously researched porous particles
made by traditional manufacturing techniques. In one formulation, drug
and excipient are present in a one-to-one ratio in a solid dispersion.
SEM samples analyzed by EDX reveal a homogeneous dispersion of tacrolimus
and lactose, indicated by the presence of nitrogen. Other studies have
produced amorphous powders with TFF and have shown through x-ray
diffraction (XRD) patterns and differential scanning calorimetry (DSC)
that these dispersions often form solid solutions.

[0209]For effective delivery of respirable low-density microparticles, a
passive inhalation device with the ability to produce high shear
velocities is required. Fortunately, most device designs already require
turbulent, high shear airflow to provide adequate force for the
separation of micronized drug from carrier lactose. The HANDIHALER®,
a single dose capsule-based DPI, was chosen for aerosolization of brittle
matrices in this study. Through a patient induced pressure drop, contents
of a size 3 capsule within the device are released by flow within and
around the capsule. Prior to discussion of further formulation
considerations, aerosol performance dependence on capsule composition is
first investigated. HPMC capsules produced a significant improvement
(P<0.05) in FPF over that of gelatin capsules while MMAD was
unchanged. The shape and area of the puncture hole created could
influence the velocity/turbidity of air entering and leaving the capsule.
In previous reports of puncture shape of gelatin and HPMC capsules, it
was concluded that more irregularly shaped holes were formed in the less
brittle HPMC capsules, relative to gelatin. For delivery of this
formulation, a smaller, non-spherical puncture may provide a greater
shear force than a large spherical opening, imparting for fracture of
friable matrices. Other non-aerodynamic advantages of powders released
from HPMC capsules include low moisture content and increased stability
at elevated humidity.

[0210]Determination of friability of brittle matrix formulations was
performed by comparing geometric particle distribution of low-density
particles emitted from the DPI device with that of "bulk" or
non-aerosolized matrices. The effect of shearing induced by the
HANDIHALER® was substantial as indicated by the difference between
the volume moment mean (d4,3) of bulk (502.4 μm) and DPI emitted
(62.0 μm) particles. The volume moment mean is a numerical
representation of the "center of gravity" of a volumetric distribution,
also known as the De Brouckere mean diameter. Because particle fracture
is vital to the aerodynamic performance of these particles, excipient
selection focusing on material properties such as strength, brittleness,
and hygroscopicity is critical. The ability to fracture the bulk
particles with air flow is consistent with the fracture of large open
friable flocs of similar particles produced by TFF, in which shear was
produced by a hydrofluoralkane in a pMDI. In each case, the shear
produces particles with proper aerodynamic and geometric diameters to
achieve high fine particle fractions. A major difference for the pMDI
approach is that the particles collapse as the HFA droplets evaporate. An
additional caveat to formulation of dry powder for inhalation is that
these excipients be nontoxic and non-irritating for delivery to the lungs
or otherwise generally recognized as safe (GRAS) by the FDA.

[0211]The influence of pharmaceutical sugars on aerosol performance was
determined by measurement of both geometric and aerodynamic properties.
In this pulmonary delivery platform, the fundamental principle for
producing highly respirable microparticles relies on the brittle fracture
of ultra low-density matrices to create small diameter particles of the
same structure and density. Accordingly, pharmaceutical materials shown
to experience brittle fracture under applied stress were chosen, such as
those used in direct compression (DC) tabletting. Sacchirides used for DC
are more likely to experience brittle fracture than ductile cellulose
excipients, and are more appropriate for our application. In addition,
some sacchirides are established as being non-irritating in the lungs.
Two sacchirides, α-lactose and raffinose, were selected based on
their brittle properties; however, the ability to induce brittle fracture
with a passive DPI device had not been determined. Mannitol, a less
hygroscopic sugar alcohol, was also selected for evaluation as an
excipient in brittle matrix powders. After production, initial visual
observations of unpackaged product showed that the skeletal structure of
TACMAN and TAC were less susceptible to ambient humidity than other
formulations.

[0212]Aerodynamic evaluation of emitted low-density microparticles on a
stage coated NGI showed elevated TED and FPF when compared to traditional
dry powder inhalation formulations (2). Initial testing of newly prepared
formulation revealed TACLAC and TACRAF as the most efficiently performing
aerosols, with FPF of 70.3 and 63.5%, respectively (Table 3).
Distribution of deposition on within the NGI, shown in FIG. 5a reveals a
lower stage deposition of TACLAC and TACRAF in comparison with the other
formulations. Assuming that all formulations have similar density, it
could be concluded that increased particle fracture of particles
containing anhydrous a-lactose and anhydrous raffinose resulted in
improved aerodynamic properties. Although some drawbacks to anhydrous
material exists (as will be discussed), complete water removal from
sacchirides often results in an significant increase in friability and
brittleness (38, 39). Specifically, anhydrous raffinose is noted for its
friability and has been determined to be the "most fragile"
pharmaceutical sugar (38). It is interesting to note that the more
brittle, anhydrous form of raffinose is also amorphous, contrasting with
the general conception that amorphous sugars are more ductile. Differing
from raffinose, anhydrous α-lactose is similar to others excipients
in that the amorphous form is commonly less brittle than the crystalline.
High TED for all formulated powders is indicative of the reduced surface
cohesion of low-density powders, normally caused by van der Waals,
capillary, and electrostatic forces in traditional formulations;
although, further analysis shows that these forces do still play a role
in particle dispersion.

[0213]Humidity had an inhibitory effect on the performance of TACLAC, most
likely due to increased plasticity of the brittle matrix. TACMAN proved
to benefit from additional moisture, as shown by an increase in FPF,
perhaps due to reduction in electrostatic charging. FIG. 6b shows the
bimodal distribution indicative of electrostatic adhesion of TACMAN at
low RH. It can also be seen that TED decreased slightly in every 50% RH
formulation, which could be expected due added formulation adhesion to
the capsule wall in the presence of moisture. Water sorption to powder
surfaces can both improve and hinder aerosol dispensability. Previous
reports have shown that dry powder formulations stored at approximately
60% RH maximize the drug FPF (40). In general, humidities >60% result
in capillary forces predominating, while electrostatic charge remains
low. Relative humidities <60% will cause elevated electrostatic
adhesion of powders due to the lack of moisture-induced charge
dissipation. For brittle matrices, presuming they are amorphous, the
plasticizing effect of water must also be considered. Amorphous materials
are particularly susceptible to water plasticization, as is the case for
anhydrous lactose and raffinose, which will result in reduced brittle
fracture and could lead to increased particle density due collapse of the
matrix structure.

[0214]Bulk and tap density testing, as defined by the USP, were used to
characterize density of each powder formulation. While all densities
measured were extremely low, the bulk density of

[0215]TACLAC was approximately twice that of the other formulations, most
likely due to matrix water absorption and subsequent particle
contraction. Tap density was also measured and used to calculate Carr's
index. Can's index, or compressibility index, is used to describe a
ductile material that undergoes plastic deformation or a brittle material
that fractures under an applied force. Assuming that all changes in
powder density were due to brittle fracture, this data provided another
indication that TACRAF is the most brittle of the powders investigated,
showing a Can's index of 50.

[0216]For comparison with USP density testing, correlation between size
distribution data produced by cascade impaction (NGI) and laser
diffraction (SPRAYTEC®) analysis were also used to determine
microparticle density. Knowing both the MMAD and the volumetric median
diameter (D.sub.[50]), equation 1 was used to calculate the skeletal
density of the sheared microparticles exiting the DPI. Approximation of
the shape factor was necessary due to its effect on aerodynamic diameter,
and was assumed to be 1.5. SEM images portrayed a jagged and irregular
morphology of the aerosolized particles, similar that of a sand particle,
which has a dynamic shape factor of 1.57. Calculation of particle density
proved to be slightly lower that measured by bulk density testing;
however, comparing formulations to one another showed a similar
relationship. It is possible that a lower prediction based on emitted
aerosols was due to non-emitted particles remaining in the device that
were excluded from characterization. Relative to each other, TACMAN and
TAC produced the lowest density particles, most likely due to their
non-hygroscopic nature, while TACRAF, and particularly TACLAC, showed
higher density. Changes in skeletal densities of lactose, and perhaps
raffinose, upon exposure to ambient moisture are due to their tendency to
adsorb water and could be explained by two mechanisms. It is likely that
adsorbed water effectively plasticizes the fragile matrix causing
lowering of the glass transition temperature (Tg) and relaxation of
supporting structures, subsequent collapsing the particle. Increased
mobility of amorphous material will also lead to formation of a more
thermodynamically stable, crystalline form. Powder collapse due to low
material Tg has been observed previously in sucrose formulations,
where inclusion of Dextran-40 significantly increased the Tg and
resulted in improved structural integrity and longer stability. By
increasing Tg of the matrix material, molecular mobility would be
limited resulting in reduction of particle shrinkage and crystal
formation. Another possibility exists for particle collapse at high
humidity (<65%) where the material becomes deliquescent, partially
dissolving in adsorbed moisture and effecting the integrity of the
particle. It is doubtful, however, that deliquescent dissolving of
lactose is a viable cause for particle collapse since the critical
relative humidity needed for this to occur is 99% RH. Mannitol and
tacrolimus, being non-hygroscopic and hydrophobic, respectively, do not
experience noticeable changes in skeletal density over time due to less
water adsorption and a higher Tg.

[0217]Inhalation of low-density microparticles formed from brittle
matrices with a marketed DPI device is a viable platform for highly
efficient deep lung delivery of drugs. Unlike delivery strategies that
utilize preformed particles, the brittle matrix TFF powders are sheared
into extremely low-density (0.05-0.01 g/cm3) microparticles in situ
by patient inspiration. After incorporation of biocompatible materials
such as pharmaceutical sugars into the formulations, aerosolization of
the resulting brittle matrices produced fine particle fractions (FPF) as
high as 70.3% and total emitted doses (TED) consistently higher than 95%.

[0218]Additional benefits of this platform for inhalation therapeutics
include solubility enhancement for amorphous particles, rapid dissolution
for high surface area sub-500 nm primary structures, and the ability to
formulate process-sensitive actives with TFF. Future studies focusing on
dose consistency, in vivo characterization, and process scale up will be
investigated to determine the viability of this platform as an
alternative to large porous particles and traditional carrier-based
formulation

[0219]The preparation of particles and respirable aggregates using a
controlled precipitation (CP) method includes a batch controlled
precipitation process. An aliquot of 1.77 grams of Brij 98 is dissolved
in 148.33 grams of deionized water. The aqueous solution is then
recirculated, using a centrifugal pump (Cole-Parmer Model 75225-10) at
maximum pump speed (9000 rpm), through recirculation loop and through
heat exchanger (Exergy Inc. Model 00283-01, series heat exchanger) until
the aqueous temperature is 5° C. An aliquot of 30.19 grams of a
solution containing 5 wt % ITZ in 1,3-dioxolane is added into the
recirculating aqueous solution over about seconds, which results in the
controlled precipitation of a particle slurry. The particle size of the
particle slurry is measured, without filtration or sonication, using a
Coulter LS 230. The particle slurry is then fed to a wiped-film
evaporator having a jacket temperature of 40° C., an absolute
pressure of 8 mm Hg, and a feed rate of 15 mL/min. The particle size of
the solvent-stripped slurry is measured, without filtration or
sonication, using a Coulter LS 230.

[0222]It is contemplated that any embodiment discussed in this
specification can be implemented with respect to any method, kit,
reagent, or composition of the invention, and vice versa. Furthermore,
compositions of the invention can be used to achieve methods of the
invention.

[0223]It will be understood that particular embodiments described herein
are shown by way of illustration and not as limitations of the invention.
The principal features of this invention can be employed in various
embodiments without departing from the scope of the invention. Those
skilled in the art will recognize, or be able to ascertain using no more
than routine experimentation, numerous equivalents to the specific
procedures described herein. Such equivalents are considered to be within
the scope of this invention and are covered by the claims.

[0224]All publications and patent applications mentioned in the
specification are indicative of the level of skill of those skilled in
the art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same extent as
if each individual publication or patent application was specifically and
individually indicated to be incorporated by reference.

[0225]The use of the word "a" or "an" when used in conjunction with the
term "comprising" in the claims and/or the specification may mean "one,"
but it is also consistent with the meaning of "one or more," "at least
one," and "one or more than one." The use of the term "or" in the claims
is used to mean "and/or" unless explicitly indicated to refer to
alternatives only or the alternatives are mutually exclusive, although
the disclosure supports a definition that refers to only alternatives and
"and/or." Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for the
device, the method being employed to determine the value, or the
variation that exists among the study subjects.

[0226]As used in this specification and claim(s), the words "comprising"
(and any form of comprising, such as "comprise" and "comprises"),
"having" (and any form of having, such as "have" and "has"), "including"
(and any form of including, such as "includes" and "include") or
"containing" (and any form of containing, such as "contains" and
"contain") are inclusive or open-ended and do not exclude additional,
unrecited elements or method steps.

[0227]The term "or combinations thereof" as used herein refers to all
permutations and combinations of the listed items preceding the term. For
example, "A, B, C, or combinations thereof" is intended to include at
least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a
particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
Continuing with this example, expressly included are combinations that
contain repeats of one or more item or term, such as BB, AAA, MB, BBC,
AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will
understand that typically there is no limit on the number of items or
terms in any combination, unless otherwise apparent from the context.

[0228]All of the compositions and/or methods disclosed and claimed herein
can be made and executed without undue experimentation in light of the
present disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be applied to
the compositions and/or methods and in the steps or in the sequence of
steps of the method described herein without departing from the concept,
spirit and scope of the invention. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by the
appended claims.

[0259][30] I. Gonda, Development of a systematic theory of suspension
inhalation aerosols. I. A framework to study the effects of aggregation
on the aerodynamic behavior of drug particles, Int. J. Pharm. 27 (1985)
99-116.